UNIVERSITY OF PISA
Research Doctorate School in
BIOLOGICAL AND MOLECULAR SCIENCES
Course in
MOLECULAR AND EXPERIMENTAL ONCOLOGY
SSD: MED/06
XXVI CYCLE (2011-‐2013)
THESIS TITLE:
PREDICTION OF DISEASE OUTCOME
IN GLIOBLASTOMA PATIENTS BY
MOLECULAR ANALYSIS
Candidate: Sara Franceschi
Supervisor: Prof. Generoso Bevilacqua
Tutor: Dr. Katia Zavaglia
Index
Abstract ... III
Introduction ... 1
1.1 The Central Nervous System ... 1
1.1.1 Neurons ... 2
1.1.2 Glia ... 3
1.2 Tumors of the CNS ... 5
1.2.1 WHO classification of Brain Tumors ... 6
1.2.2 Astrocytic Tumors ... 8
1.3 Glioblastoma ... 10
1.3.1 Clinical features ... 11
1.3.2 Macroscopy ... 12
1.3.3 Spread and metastasis ... 13
1.3.4 Primary and secondary glioblastoma ... 14
1.3.5 Histopathology ... 17
1.3.6 Tumor progression and aggressive behavior ... 20
1.3.7 Molecular Pathogenesis ... 23
1.3.8 Treatment and prognostic factors ... 31
1.4 TCGA molecular subtypes of glioblastoma ... 36
1.5 MicroRNAs in Glioblastoma ... 40
1.6 Fusion Genes ... 42
Aim ... 44
Materials and Methods ... 46
3.1 Glioma sample database ... 46
3.2 Analysis setup ... 46
3.2.1 Italian gliomas samples collection ... 46
3.2.1.1 Analysis of 15 molecular markers panel involved both in glioma tumor progression and radiotherapy response ... 47
3.2.1.2 PCR Array analysis and time of first recurrence correlation ... 47
3.2.1.3 RNA-‐Seq analysis and correlation with time of first recurrence .. 47
3.2.2 American GMB samples collection and Nanostring analysis ... 48
3.3 DNA extraction ... 48
3.4 Pyrosequencing analysis ... 48
3.4.1 Mutational and genotyping SNPs analysis ... 50
3.4.2 Methylation analysis ... 50
3.5 Copy number variation analysis ... 50
3.6 Codeletion analysis ... 51
3.7 RNA extraction ... 51
3.9 Ion Proton Technology for RNA-‐seq ... 54
3.9.1 Library preparation ... 55
3.9.2 Template preparation ... 57
3.9.3 Sequencing on the Ion Proton ... 58
3.9.4 Mapping of RNA-‐Seq reads using TopHat 2 ... 58
3.9.5 Transcript assembly and differential analysis with Cuffmerge and Cuffdiff ... 59
3.9.6 Gene Ontology annotation and molecular networks design ... 60
3.9.7 Fusion Trancripts analysis ... 60
3.10 Nanostring analysis ... 60
3.11 Statistical analysis ... 63
Results ... 64
4.1 Analysis of 15 molecular markers involved both in glioma tumor progression and radiotherapy response ... 64
4.1.1 WHO IV grade samples molecular markers alterations frequency 64 4.1.2 WHO IV grade molecular marker alteration association ... 66
4.1.3 Lower grade glioma samples molecular alterations frequency ... 67
4.1.4 Lower grade and grade IV gliomas molecular alterations comparison ... 67
4.2 PCR Array analysis and time of first recurrence correlation .. 69
4.3 RNA-‐Seq analysis ... 70
4.3.1 Gene expression level differences related to time of first recurrence ... 70
4.3.2 Fusion gene transcripts ... 73
4.3.2.1 Fusion gene transcripts related to the time of first recurrence .... 73
4.3.2.2 Gene transcript fusions frequencies in GBM ... 73
4.4 Nanostring Analysis and outcome correlation with TCGA molecular classification ... 74
4.4.1 TCGA molecular classification and time of first recurrence ... 75
4.4.2 TCGA molecular classification and survival after Bevacizumab therapy ... 82
4.4.3 TCGA molecular classification and overall survival (OS) ... 85
4.4.4 TCGA molecular classification and therapy response ... 90
4.4.4.1 1st and 2nd line therapies with the same survival response ... 91
4.4.4.2 1st and 2nd line therapies with completely different survival response ... 93
Discussion ... 96
Conclusions ... 104
Future Prespectives ... 105
Abstract
Malignant gliomas, especially glioblastoma (GBM), represent the most common primary malignant tumors of the adult central nervous system. Surgical resection, radiotherapy and adjuvant chemotherapy are currently the standard of care for GBM. However, despite progress in recent years, due to the infiltrative and dispersive nature of the tumor, recurrence rate remains high and typically results in very poor prognosis. From a practical perspective, controlling growth and dispersal of the recurrence may have a greater impact on disease-‐free survival. Our study had the intent to provide novel information on GBM tumor aggressive behavior by connecting multiple molecular markers, that could play an important role in tumor progression, with the outcome of the patients meant as the time of recurrence, time of survival after a specific therapy and overall survival. In this work we collected a total of 205 formalin fixed paraffin embedded (FFPE) first-‐surgery glioma samples from two different populations, Italian and American.
Italian glioma samples were molecularly characterized investigating 15 of the most studied candidate molecular markers involved both in glioma tumor progression and radiotherapy response : IDH1 and IDH2 mutations, SNPs (XRCC1, XRCC3, RAD51, GSTP1), EGFR and ERBB2 amplification, deletion of PTEN, TP53 and CDK2NA genes, presence of the active mutant form of EGFR (EGFRvIII), MGMT promoter methylation and 1p19q codeletion. To better understand the dynamics of genomic alterations associated with GBM recurrence in more detail we selected 19 GBM samples with specific recurrence free survival outcome; by using real-‐time PCR we profiled the expression of 84 genes important for cell-‐cell and cell-‐matrix interactions, the expression of 84 miRNAs known to alter their expression during nervous system-‐related carcinogenesis and a copy number variation analysis of 23 genes reported to undergo frequent genomic alterations in human glioma tumor DNA. In order to have even more selected samples with differences in time of first recurrence we
chosen 6 GBM; on those samples we performed a whole transcriptome sequencing analyses (RNA-‐seq) by using Ion Proton System technology.
American GBM samples were analyzed using Nanostring technology. We used a customized gene panel to evaluate the expression levels of 151 genes involved in glioblastoma and classified our samples into one of the four TCGA molecular classes: Classical, Mesenchymal, Neural and Proneural.
We found strong correlation between expression levels of different genes related to cellular migration and invasion and patient outcome. Those genes had also high power to predict patient outcome on the basis of the combination of their expression levels. By using RNA-‐seq, we found several genes with statistically significant expression level differences in GBM patient with distinct outcomes, and we were able to identify specific molecular functions networks into those groups of tumors. Moreover we were able to identify specific gene fusion transcripts with high probability of a “driver” behavior in the oncogenic process; we also detected exclusively gene fusion transcripts in GBM patient with distinct outcomes.
Furthermore we found strong correlation between TCGA molecular classification and patient outcome and we selected characteristic genes with expression levels related both to the molecular TCGA subclass and the survival.
In conclusion our study shows how the integration of miRNA and mRNA espression and genomic alterations can define a GBM molecular profile related to patient outcome. Investigation of diagnostic and prognostic value of these molecular markers could be useful to allow better predictions of GBM patients prognosis and response to therapies.
1
Introduction
1.1 The Central Nervous System
The human central nervous system (CNS) is an enormously complex tissue serving the organism as a processing center linking information between the outside world and the body. The principal functional unit of the CNS is the neuron; the best estimates are that there are about 1011 neurons in the human
brain. Neurons, although similar in many ways to other cells in the body, are unique in their ability to receive, store, and transmit information. Neurons differ greatly from one another in many important properties: their functional roles (sensory, motor, autonomic), the distribution of their connections, the neurotransmitters they use for synaptic transmission, their metabolic requirements, and their levels of electrical activity at a given moment. A set of neurons, not necessarily clustered together in a region of the brain, may thus be singled out for destruction in a pathologic condition (selective vulnerability) because it shares one or more of these properties. Furthermore, and of particular importance in medicine, most mature neurons are postmitotic cells that are incapable of cell division, so destruction of even a small number of neurons responsible for a specific function may leave the patient with a severe clinical neurologic deficit. In comparison to other organ systems of the body, the nervous system has several unique anatomic and physiologic characteristics: the protective bony enclosure of the skull and spinal column that contains it, a specialized system of autoregulation of cerebral blood flow, metabolic substrate requirements, the absence of a conventional lymphatic system, a special cerebrospinal fluid (CSF) circulation, limited immunologic surveillance, and distinctive responses to injury and wound healing. As a result of these special
characteristics, the CNS is vulnerable to unique pathologic processes, and the reactions of CNS tissue to injury differ considerably from those encountered elsewhere [1,2]. The principal cells of the CNS are neurons, glia, and the cells that compose the meninges and blood vessels.
1.1.1 Neurons
In the CNS, neurons are topographically organized either as aggregates (nuclei, ganglia) or as elongated columns or layers (such as the intermediolateral gray column of the spinal cord or the six-‐layered cerebral cortex)[3]. Functional domains are located in many of these anatomically defined regions (such as the hypoglossal nucleus of the medulla for motor fibers of the twelfth cranial nerve; calcarine cortex of the occipital lobe for primary visual cortex). In addition, as a further dimension of anatomic functional specificity, some cortical and subcortical neurons and their projections are arranged somatotopically (such as motor and sensory homunculi)[4].
Neurons vary considerably in structure and size throughout the nervous system and within a given brain region. With conventional histologic preparations, an anterior horn neuron in the spinal cord has a cell body (perikaryon) that is about 50 µm wide, a relatively large and somewhat eccentrically placed nucleus, a prominent nucleolus, and abundant Nissl substance; the nucleus of a granule cell neuron of the cerebellar cortex is about 10 µm across, and its perikaryon and nucleolus are not readily visible by light microscopy. Electron microscopic study reveals further variability among neurons in cytoplasmic content and the shape of the cells and their processes [5]. Characteristic ultrastructural features common to many neurons include microtubules, neurofilaments, prominent Golgi apparatus and rough endoplasmic reticulum, and synaptic specializations. Despite these shared structures, axon length may vary greatly (hundreds of microns for interneurons versus a meter for an upper motor neuron). Immunohistochemical markers for neurons and their processes commonly used in diagnostic work include neurofilament protein, NeuN, and synaptophysin[6].
1.1.2 Glia
Glial cells are derived from neuroectoderm (macroglia: astrocytes, oligodendrocytes, ependyma) or from bone marrow (microglia). Glial cells have important structural and metabolic interactions with neurons and their dendritic and axonal processes; they also have a primary role in a wide range of normal functions and reactions to injury, including inflammation, repair, fluid balance, and energy metabolism. The size and shape of the nucleus helps in the light microscopic distinction of one glial cell type from another, as their cytoplasmic processes are often not apparent on H&E preparations and can be demonstrated only with the use of metallic impregnation, immunohistochemical, or electron microscopic methods. Astrocytes typically have round to oval nuclei (10 µm wide) with evenly dispersed, pale chromatin; oligodendrocytes have a denser, more homogeneous chromatin in a rounder and smaller nucleus (8 µm); and microglia have an elongated, irregularly shaped nucleus (5 to 10 µm) with clumped chromatin. Ependymal cells, on the other hand, do have visible cytoplasm; seen with H & E, they are columnar epithelial-‐like cells with a ciliated/microvillous border facing the ventricular surface with pale, vesiculated nuclei (each about 8 µm) located at the abluminal end of the cell.
Astrocytes. This glial cell is found throughout the CNS in both gray and white
matter. Protoplasmic astrocytes occur mainly in the gray matter; fibrous astrocytes occur in white and gray matter. The cell derives its name from its star-‐shaped appearance, which is imparted by the multipolar, branching cytoplasmic processes that emanate from the cell body containing the characteristic cytoplasmic intermediate filament protein called glial fibrillary acidic protein (GFAP). These are seen well in tissue sections only with metallic impregnation techniques (for example the Golgi method) or immunohistochemical preparations (Figure 1.1).
Figure 1.1 A, Astrocytes and their processes. Some processes extend toward
blood vessels. B, Immunoperoxidase staining for glial fibrillary acidic protein shows astrocytic perinuclear cytoplasm and well-‐developed processes (brown)[7].
The filaments are either aggregated in fascicles (in protoplasmic astrocytes) or dispersed diffusely throughout the cytoplasm (in fibrous astrocytes). Some astrocytic processes are directed toward neurons and their processes and synapses, where they are believed to act as metabolic buffers or detoxifiers, suppliers of nutrients, and electrical insulators. Others surround capillaries or extend to the subpial and subependymal zones, where they contribute to barrier functions controlling the flow of macromolecules between the blood, the CSF, and the brain. Astrocytes are also the principal cells responsible for repair and scar formation in the brain. Fibroblasts, which have a major role in wound healing elsewhere, are located mainly around large CNS blood vessels and in the meninges; they participate in wound healing only to a limited extent (primarily in the organization of subdural hematomas and the formation of abscess cavities).
Oligodendrocytes. Oligodendroglial cytoplasmic processes wrap around the
axons of neurons to form myelin in a manner analogous to the Schwann cells of the peripheral nervous system. Unlike Schwann cells, which form the myelin of a single internode, each oligodendrocyte myelinates numerous internodes on multiple axons. In routine sections, oligodendroglia are recognizable by their small, rounded, lymphocyte-‐like nuclei, often in linear arrays. Injury to oligodendroglial cells is a feature of acquired demyelinating disorders (for example, multiple sclerosis); it is also seen in the leukodystrophies.
Oligodendroglial nuclei may harbor viral inclusions in progressive multifocal leukoencephalopathy.
Ependymal Cells. Ependymal cells line the ventricular system. They are closely
related to the cuboidal cells comprising the choroid plexus. Disruption of ependymal cells is often associated with a local proliferation of subependymal astrocytes to produce small irregularities on the ventricular surfaces termed ependymal granulations. Certain infectious agents, particularly cytomegalovirus, may produce extensive ependymal injury, and viral inclusions may be seen within ependymal cells.
Microglia. Microglia are mesoderm-‐derived cells whose primary function is to
serve as a fixed macrophage system in the CNS. They express many surface markers in common with peripheral monocytes/macrophages (such as CR3 and CD68). They respond to injury by (a) proliferation; (b) developing elongated nuclei (rod cells), as in neurosyphilis; (c) forming aggregates about small foci of tissue necrosis (microglial nodules); or (d) congregating around cell bodies of dying neurons (neuronophagia). In addition to resident microglia, blood-‐ derived macrophages are the principal phagocytic cells present in inflammatory foci [8,9,10].
1.2 Tumors of the CNS
The annual incidence of tumors of the CNS ranges from 10 to 17 per 100,000 persons for intracranial tumors and 1 to 2 per 100,000 persons for intraspinal tumors; about half to three-‐quarters are primary tumors, and the rest are metastatic.
Tumors of the nervous system have several unique characteristics that set them apart from neoplastic processes elsewhere in the body. First, the distinction between benign and malignant lesions is less evident in the CNS than in other organs. Some glial tumors with histologic features of a benign neoplasm, including low mitotic rate, cellular uniformity, and slow growth, may infiltrate large regions of the brain, thereby leading to serious clinical deficits and poor prognosis. Second, the ability to surgically resect infiltrating glial neoplasms without compromising neurologic function is limited. Third, the anatomic site of the neoplasm can have lethal consequences irrespective of histologic
classification; for example, a benign meningioma, by compressing the medulla, can cause cardiorespiratory arrest. Finally, the pattern of spread of primary CNS neoplasms differs from that of other tumors: even the most highly malignant gliomas rarely metastasize outside the CNS. The subarachnoid space provides a pathway for spread, so seeding along the brain and spinal cord can occur in highly anaplastic as well as in well-‐differentiated neoplasms that extend into the CSF pathways.
1.2.1 WHO classification of Brain Tumors
Histological grading is a means of predicting the biological behavior of a neoplasm. In the clinical setting, tumor grade is a key factor influencing the choice of therapies, particularly determining the use of adjuvant radiation and specific chemotherapy protocols. Since its first publication in 1979, the WHO Classification of Tumors of the Nervous System has included a grading scheme that is a “malignancy scale” ranging across a wide variety of neoplasms rather than a strict histological grading system [11,12].
WHO grading is widely used, having incorporated or largely replaced other previously published grading systems. Although it is not a requirement for application of the WHO classification for some tumors, including gliomas and meningiomas, numerical WHO grades are useful additions to the diagnosis. Grade I lesions generally include tumors with low proliferative potential and the possibility of cure following surgical resection alone. Lesions designated grade II are generally infiltrative in nature and, despite low level proliferative activity, often recur. Some type II tumors tend to progress to higher grades of malignancy, for example, low-‐grade diffuse astrocytomas that transform to anaplastic astrocytoma and glioblastoma. Similar transformation occurs in oligodendroglioma and mixed gliomas. The designation grade III is generally reserved for lesions with histological evidence of malignancy, including nuclear atypia and brisk mitotic activity. In most settings, patients with grade III tumors receive adjuvant radiation and/or chemotherapy. The designation grade IV is assigned to cytologically malignant, mitotically active, necrosis-‐prone neoplasms often associated with rapid pre-‐ and postoperative disease evolution
and a fatal outcome. Examples of grade IV neoplasms include glioblastoma, most embryonal neoplasms and many sarcomas as well.
Grading has been systematically evaluated and successfully applied to a spectrum of diffusely infiltrative astrocytic tumors. These neoplasms are graded in a three-‐tiered system similar to that of the Ringertz [13], St. Anne-‐Mayo [14] and the previously published WHO schemes[15].
As currently defined by the WHO, tumors with cytological atypia alone are considered grade II (diffuse astrocytoma), those also showing anaplasia and mitotic activity are considered grade III (anaplastic astrocytoma), and tumors additionally showing microvascular proliferation and/or necrosis are WHO grade IV (Table 1.1).
Atypia is defined as variation in nuclear shape or size with accompanying hyperchromasia. Mitoses must be unequivocal, but no special recognition is given to their number or morphology. Since the finding of a solitary mitosis in an ample specimen does not confer grade III behavior, separation of grade II from grade III tumors may be more reliably achieved by determination of MIB-‐1 labelling indices [16,17,18].
Endothelial proliferation is defined as apparent multi-‐layering of endothelium, rather than simple hypervascularity, or glomeruloid vasculature. Necrosis may be of any type; perinecrotic palisading need not be present. Simple apposition of cellular zones with intervening pallor suggestive of incipient necrosis is insufficient.
WHO grade is one component of a combination of criteria used to predict a response to therapy and outcome. Other criteria include clinical findings, such as age of the patient, performance status and tumor location; radiological features such as contrast enhancement; extent of surgical resection; proliferation indices; and genetic alterations. For each tumor entity, combinations of these parameters contribute to an overall estimate of prognosis. Despite these variables, patients with WHO grade II tumors typically survive more than 5 years, and those with grade III tumors survive 2-‐3 years. The prognosis of patients with WHO grade IV tumors depends largely upon whether effective treatment regimens are available. The majority of
glioblastoma patients, particularly the elderly, succumb to the disease within a year.
11
II IIII IIIIII IIVV A
Assttrrooccyyttiicc ttuummoouurrss Subependymal giant cell
astrocytoma • Pilocytic astrocytoma • Pilomyxoid astrocytoma • Diffuse astrocytoma • Pleomorphic xanthoastrocytoma • Anaplastic astrocytoma • Glioblastoma •
Giant cell glioblastoma •
Gliosarcoma •
O
Olliiggooddeennddrroogglliiaall ttuummoouurrss
Oligodendroglioma •
Anaplastic oligodendroglioma • O
Olliiggooaassttrrooccyyttiicc ttuummoouurrss
Oligoastrocytoma •
Anaplastic oligoastrocytoma •
EEppeennddyymmaall ttuummoouurrss
Subependymoma •
Myxopapillary ependymoma •
Ependymoma •
Anaplastic ependymoma •
C
Chhoorrooiidd pplleexxuuss ttuummoouurrss
Choroid plexus papilloma • Atypical choroid plexus papilloma •
Choroid plexus carcinoma •
O
Otthheerr nneeuurrooeeppiitthheelliiaall ttuummoouurrss
Angiocentric glioma •
Chordoid glioma of
the third ventricle •
N
Neeuurroonnaall aanndd mmiixxeedd nneeuurroonnaall--gglliiaall ttuummoouurrss
Gangliocytoma •
Ganglioglioma •
Anaplastic ganglioglioma •
Desmoplastic infantile astrocytoma
and ganglioglioma •
Dysembryoplastic
neuroepithelial tumour •
II IIII IIIIII IIVV
Central neurocytoma •
Extraventricular neurocytoma • Cerebellar liponeurocytoma • Paraganglioma of the spinal cord • Papillary glioneuronal tumour • Rosette-forming glioneuronal
tumour of the fourth ventricle • PPiinneeaall ttuummoouurrss
Pineocytoma •
Pineal parenchymal tumour of
intermediate differentiation • •
Pineoblastoma •
Papillary tumour of the pineal region • • EEmmbbrryyoonnaall ttuummoouurrss
Medulloblastoma •
CNS primitive neuroectodermal
tumour (PNET) •
Atypical teratoid / rhabdoid tumour • TTuummoouurrss ooff tthhee ccrraanniiaall aanndd ppaarraassppiinnaall nneerrvveess
Schwannoma •
Neurofibroma •
Perineurioma • • •
Malignant peripheral nerve
sheath tumour (MPNST) • • •
M
Meenniinnggeeaall ttuummoouurrss
Meningioma •
Atypical meningioma •
Anaplastic / malignant meningioma •
Haemangiopericytoma •
Anaplastic haemangiopericytoma •
Haemangioblastoma •
TTuummoouurrss ooff tthhee sseellllaarr rreeggiioonn
Craniopharyngioma •
Granular cell tumour
of the neurohypophysis •
Pituicytoma •
Spindle cell oncocytoma
of the adenohypophysis •
WHO grades of CNS tumours
Table 1.1 WHO grades of CNS tumors [19].
1.2.2 Astrocytic Tumors
Pilocytic astrocytoma (WHO grade I) is relatively circumscribed, slowly growing, often cystic astrocytoma occurring in children and young adults,
histologically characterized by a biphasic pattern with varying proportions of compacted bipolar cells associated with Rosenthal fibers and loose-‐textured multipolar cells associated with microcysts and eosinophilic granular bodies/hyaline droplets.
Subependymal giant cell astrocytoma (WHO grade I) is a benign, slowly growing tumor typically arising in the wall of the lateral ventricles and composed of large ganglioid astrocytes.
Pleomorphic xanthoastrocytoma (WHO grade II) is an astrocytic neoplasm with a relatively favourable prognosis, typically encountered in children and young adults, with superficial location in the cerebral hemispheres and involvement of the meninges; characteristic histological features include pleomorphic and lipidized cells expressing GFAP and often surrounded by a reticulin network as well as eosinophilic granular bodies.
Diffuse astrocytoma (WHO grade II) is a diffusely infiltrating astrocytoma that typically affects young adults and is characterized by a high degree of cellular differentiation and slow growth; the tumor occurs throughout the CNS but is preferentially located supratentorially and has an intrinsic tendency for malignant progression to anaplastic astrocytoma and, ultimately, glioblastoma. Anaplastic astrocytoma (WHO grade III) is a diffusely infiltrating malignant astrocytoma that primarily affects adults, is preferentially located in the cerebral hemispheres, and is histologically characterized by nuclear atypia, increased cellularity and significant proliferative activity. The tumor may arise from diffuse astrocytoma WHO grade II or de novo, without evidence of a less malignant precursor lesion, and has an inherent tendency to undergo progression to glioblastoma.
Glioblastoma (WHO grade IV) is the most frequent primary brain tumor and the most malignant neoplasm with predominant astrocytic differentiation; histopathological features include nuclear atypia, cellular pleomorphism, mitotic activity, vascular thrombosis, microvascular proliferation and necrosis. It typically affects adults and is preferentially located in the cerebral hemispheres. Most glioblastomas manifest rapidly de novo, without recognizable precursor lesions (primary glioblastoma). Secondary glioblastomas develop slowly from diffuse astrocytoma WHO grade II or
anaplastic astrocytoma (WHO grade III). Due to their invasive nature, glioblastomas cannot be completely resected, and despite progress in radio/chemotherapy, less than half of patients survive more than a year, with older age as the most significant adverse prognostic factor.
Gliomatosis cerebri is a diffuse glioma (usually astrocytic) growth pattern consisting of exceptionally extensive infiltration of a large region of the central nervous system, with involvement of at least three cerebral lobes, usually with bilateral involvement of the cerebral hemispheres and/or deep gray matter, and frequent extension to the brain stem, cerebellum, and even the spinal cord. Gliomatosis cerebri most commonly displays an astrocytic phenotype, although oligodendrogliomas and mixed oligoastrocytomas can also present with the gliomatosis cerebri growth pattern.
1.3 Glioblastoma
Glioblastoma is the most frequent brain tumor, accounting for approximately 12–15% of all intracranial neoplasms and 60-‐75% of astrocytic tumors [20,21]. In most European and North American countries, the incidence is in the range of 3-‐4 new cases per 100 000 population per year [20]. The incidence rate of glioblastoma in the USA, adjusted to the US Standard Population, is 2.96 new cases per 100 000 population per year [22]. The corresponding rate in a population-‐based study in Switzerland was 3.55 new cases, adjusted to the European Standard Population [21]. Glioblastoma may manifest at any age, but preferentially affects adults, with a peak incidence at between 45 and 75 years of age. In a population-‐based study in the Canton of Zurich, Switzerland, the mean age of patients with glioblastoma was 61.3 years: more than 80% of patients were older than 50 years [23] whereas only 7 out of 715 cases (1%) were diagnosed in patients younger than 20 years old. The male:female ratio of glioblastoma patients is 1.26 in USA and 1.28 in Switzerland (Figure 1.2) [21].
33 Glioblastoma
Definition
The most frequent primary brain tumour and the most malignant neoplasm with predominant astrocytic differentiation; histopathological features include nuclear atypia, cellular pleomorphism, mitotic activity, vascular thrombosis, microvascular proliferation and necrosis. It typically affects adults and is preferentially located in the cerebral hemispheres. Most
glio-blastomas manifest rapidly de novo,
without recognizable precursor lesions (primary glioblastoma). Secondary glio-blastomas develop slowly from diffuse astrocytoma WHO grade II or anaplastic astrocytoma (WHO grade III). Due to their invasive nature, glioblastomas cannot be completely resected and despite progress in radio/chemotherapy, less than half of patients survive more than a year, with older age as the most significant adverse prognostic factor.
ICD-O code 9440/3
Grading
Glioblastoma and its variants correspond to WHO grade IV.
Synonyms and historical annotation The term glioblastoma is used synony-mously with “glioblastoma multiforme”. Scherer {2013} and Kernohan {1093} were instrumental in developing the concept that glioblastoma is a malignant astrocytoma that sometimes develops by progression from a lower grade lesion.
Incidence
Glioblastoma is the most frequent brain tumour, accounting for approximately 12–15% of all intracranial neoplasms and 60-75% of astrocytic tumours {1260, 1625}. In most European and North American countries, the incidence is in the range of 3-4 new cases per 100 000 population per year {1260}. The incidence rate of glioblastoma in the USA, adjusted to the US Standard Population, is 2.96 new cases per 100 000 population per year {305}. The corresponding rate in a population-based study in Switzerland was 3.55 new cases, adjusted to the European Standard Population {1625}. Age and sex distribution
Glioblastoma may manifest at any age, but preferentially affects adults, with a peak incidence at between 45 and 75 years of age. In a population-based study in the Canton of Zurich, Switzerland, the mean age of patients with glioblastoma was 61.3 years: more than 80% of patients were older than 50 years {1620}, whereas only 7 out of 715 cases (1%) were diagnosed in patients younger than 20 years old. The male:female ratio of glioblastoma patients is 1.26 in USA and 1.28 in Switzerland {1624}.
Localization
Glioblastoma occurs most often in the subcortical white matter of the cerebral hemispheres. In a series of 987 glio-blastomas from the University Hospital
Zurich, the most frequently affected sites were the temporal (31%), parietal (24%), frontal (23%) and occipital lobes (16%). Combined fronto-temporal location is particularly typical. Tumour infiltration often extends into the adjacent cortex and through the corpus callosum into the contralateral hemisphere. Glioblastoma of the basal ganglia and thalamus is not uncommon, especially in children. Intraventricular glioblastoma is excep-tional {1281}. Glioblastoma of the brain stem (‘malignant brain stem glioma’) is infrequent and often affects children {478}. Cerebellum and spinal cord are rare sites for this neoplasm.
Clinical features
The clinical history of the disease is usually short (less than 3 months in more than 50% of cases), unless the neoplasm has developed from a lower grade astrocytoma (secondary glioblastoma). Symptoms and signs of raised intracranial pressure (for example headache, nausea/vomiting with papilledema) are common. Up to one third of patients will experience an epileptic seizure. Non-specific neuro-logical symptoms such as headache and personality changes can also occur. Macroscopy
Despite the short duration of symptoms in many cases of glioblastoma, the tumours are often surprisingly large at the time of presentation, and may occupy much of a lobe. The lesion is usually unilateral, but those in the brain stem and corpus callosum can be bilaterally symmetrical. Supratentorial bilateral extension is due to rapid growth along myelinated struc-tures, in particular across the corpus callosum and along the fornices toward the temporal lobes.
Glioblastomas are poorly delineated, the cut surface showing a variable colour with peripheral greyish tumour masses and central areas of yellowish necrosis from myelin breakdown. The peripheral hypercellular zone appears macroscopi-cally as a soft, grey rim or a grey band of tumour tissue. However, necrotic tissue
Glioblastoma
P. Kleihues P.C. Burger K.D. Aldape D.J. Brat W. Biernat D.D. Bigner Y. Nakazato K.H. Plate F. Giangaspero A. von Deimling H. Ohgaki W.K. CaveneeFig. 1.26 Age and sex distribution of glioblastoma, based on 715 cases from a population-based study, Canton of Zurich, Switzerland.
Figure 1.2 Age and sex distribution of glioblastoma, based on 715 cases from a
population-‐based study, Canton of Zurich, Switzerland [19].
Glioblastoma occurs most often in the subcortical white matter of the cerebral hemispheres. In a series of 987 glioblastomas from the University Hospital Zurich, the most frequently affected sites were the temporal (31%), parietal (24%), frontal (23%) and occipital lobes (16%) [19]. Combined fronto-‐temporal location is particularly typical. Tumor infiltration often extends into the adjacent cortex and through the corpus callosum into the contralateral hemisphere.
1.3.1 Clinical features
The clinical history of the disease is usually short (less than 3 months in more than 50% of cases), unless the neoplasm has developed from a lower grade astrocytoma (secondary glioblastoma). Symptoms and signs of raised intracranial pressure (for example headache, nausea/vomiting with papilledema) are common. Up to one third of patients will experience an epileptic seizure. Non-‐specific neurological symptoms such as headache and personality changes can also occur.
Glioblastoma has an unfavorable prognosis mainly due to its high propensity for tumor recurrence. It has been suggested that GBM recurrence is inevitable after a median survival time of 32 to 36 weeks [24].
The natural history of recurrent GBM, however, is largely undefined for the following reasons: a) lack of uniform definition and criteria for tumor recurrence; b) institutional variability in treatment philosophy; and c) the heterogeneous nature of the disease, including location of recurrence and distinct mechanisms believed to contribute to known subtypes of GBM.
Essentially all patients develop recurrent or progressive disease after initial therapy, after which the median survival is approximately 6 months [25,26]. Once a patient with GBM relapses following initial surgery, radiotherapy, and chemotherapy, salvage strategies are often limited, and survival is short. This most likely is the result of molecular and genetic changes within the tumor itself, or possibly is a consequence of prior therapies. In any event, the tumor is much more resistant to treatment, and tumor control is often short.
1.3.2 Macroscopy
Despite the short duration of symptoms in many cases of glioblastoma, the tumors are often surprisingly large at the time of presentation, and may occupy much of a lobe. The lesion is usually unilateral, but those in the brain stem and corpus callosum can be bilaterally symmetrical. Supratentorial bilateral extension is due to rapid growth along myelinated structures, in particular across the corpus callosum and along the fornices toward the temporal lobes. Glioblastomas are poorly delineated, the cut surface showing a variable colour with peripheral greyish tumor masses and central areas of yellowish necrosis from myelin breakdown. The peripheral hypercellular zone appears macroscopically as a soft, grey rim or a grey band of tumor tissue. However, necrotic tissue may also border adjacent brain structures without an intermediate zone of macroscopically detectable tumor tissue. The central necrosis may occupy as much as 80% of the total tumor mass.
Glioblastomas are typically stippled with red and brown foci of recent and remote haemorrhages. Extensive haemorrhages may occur and evoke stroke-‐ like symptoms, which are sometimes the first clinical sign of the tumor. Macroscopic cysts, when present, contain a turbid fluid and represent liquefied necrotic tumor tissue, quite in contrast to the well-‐delineated retention cysts in diffuse astrocytomas WHO grade II. Most glioblastomas of the cerebral hemispheres are clearly intraparenchymal with an epicentre in the white matter. Infrequently, they are largely superficial and in contact with the leptomeninges and dura and may be interpreted by the neuroradiologist or surgeon as metastatic carcinoma, or as an extra-‐axial lesion such as
meningioma. Cortical infiltration may produce a preserved gyriform rim of thickened grey cortex overlying a necrotic zone in the white matter (Figure 1.3).
35
Glioblastoma
that in approximately 3% of these, tumour foci vary in their histological appearance. True multifocal glioblastomas are most likely polyclonal if they occur infra- and supratentorially, i.e. outside easily accessible routes like the cerebrospinal fluid pathways or the median commissures {1977}. Multiple independently arising gliomas would, by definition, be of polyclonal origin and their existence can only be proven by application of molecular
markers which, in informative cases, allow a distinction between tumours of common or independent origin {141, 157, 1184}.
Primary and secondary glioblastoma The terms primary and secondary glioblastoma were first used by Scherer in 1940 who noted: “From a biological and clinical point of view, the secondary glioblastomas developing in
astrocy-tomas must be distinguished from ‘primary’ glioblastomas; they are probably responsible for most of the glioblastomas of long clinical duration” {2013}. The majority of glioblastomas (>90%) develop very rapidly with a short clinical history (usually <3 months), without clinical or histopathological evidence of a pre-existing, less malignant precursor lesion (primary or de novo glioblastoma). They typically develop in older patients (mean,
Fig. 1.29 Macroscopic features of glioblastoma (GBM). A Large GBM in the left frontal lobe extending into the corpus callosum and the contralateral white matter. B,C GBM of
the left fronto-temporal region with invasion of the fornices, spread to the lower horn of the left ventricle and (C) extension to the corpus callosum and adjacent parieto-occipital structures. D GBM of the left basal ganglia and spread into the right hemisphere with formation of a cystic necrosis. E GBM of the left hemisphere with extensive necrosis occupying most of the tumour mass. F Right fronto-temporal GBM with mass shifting and subfalcial herniation. G GBM with unusual intraventricular location. H Malignant brain stem GBM in a child. I GBM of the left hemisphere with extensive necrosis and spread to the right frontal lobe via the corpus callosum.
A B C
D E F
G H I
Figure 1.3 Macroscopic features of glioblastoma (GBM). A, Large GBM in the left
frontal lobe extending into the corpus callosum and the contralateral white matter. B, GBM of the left fronto-‐temporal region with invasion of the fornices, spread to the lower horn of the left ventricle and C, extension to the corpus callosum and adjacent parieto-‐occipital structures. D, GBM of the left basal ganglia and spread into the right hemisphere with formation of a cystic necrosis.
E, GBM of the left hemisphere with extensive necrosis occupying most of the
tumor mass. F, Right fronto-‐temporal GBM with mass shifting and subfalcial herniation. G, GBM with unusual intraventricular location. H, Malignant brain stem GBM in a child. I, GBM of the left hemisphere with extensive necrosis and spread to the right frontal lobe via the corpus callosum [19].
1.3.3 Spread and metastasis
Although infiltrative spread is a common feature of all diffuse astrocytic tumors, glioblastoma is particularly notorious for its rapid invasion of neighbouring brain structures [27].
A very common feature is extension of the tumor through the corpus callosum into the contralateral hemisphere, creating the image of a bilateral, symmetrical lesion (‘butterfly glioma’). Similarly, rapid spread is observed in the internal capsule, fornix, anterior commissure and optic radiation. These structures may become enlarged and distorted but continue to serve as a ‘highway’, allowing the formation of new tumor masses at their opposite projection site, thus leading to the neuroradiological image of a multifocal glioblastoma. Invading cells reside outside the contrast-‐enhancing rim of the tumor, thereby escaping surgical resection and evading high doses of radiation during radiotherapy. This likely represents the source for local recurrence usually seen after such therapy. Despite its rapid, infiltrative growth, glioblastoma tends not to invade the subarachnoidal space, and consequently rarely metastasizes via the cerebrospinal fluid [28]. A number of molecular mediators of invasion in glioblastoma have been described, including activation of the TGF-‐ß and AKT pathways [29,30]. Tumor hypoxia may also promote invasion through the activation of HIF-‐1α [31].
An important aspect of glioblastoma invasion is the elaboration of a migration-‐ enhancing extracellular matrix by tumor cells [32] as well as secretion of proteolytic enzymes which permit invasion through this matrix. Consistent with this notion are recent gene expression profiling studies that identify a subset of tumors with elevated expression of extracellular matrix components as well as intracellular proteins associated with cell motility [33,34].
The interplay between the variety of matrix and growth factor receptors as well as activation of signalling pathways which facilitate tumor cell invasion is being recognized as a composite, dynamic consequence of altered cell-‐cell adhesion, proteolytic remodelling and synthesis of ECM, as well as selective expression and activation of integrins [35]. Activation of migration (seen at the leading edge of the neoplasm) appears to be associated with a decrease in proliferation rate [36], which may have therapeutic consequences [37].
1.3.4 Primary and secondary glioblastoma
The terms primary and secondary glioblastoma were first used by Scherer in 1940 who noted: “From a biological and clinical point of view, the secondary
glioblastomas developing in astrocytomas must be distinguished from ‘primary’ glioblastomas; they are probably responsible for most of the glioblastomas of long clinical duration” [38].
The majority of glioblastomas (>90%) develop very rapidly with a short clinical history (usually <3 months), without clinical or histopathological evidence of a pre-‐existing, less malignant precursor lesion (primary or de novo glioblastoma). They typically develop in older patients (mean, 62 years) [39,40].
Glioblastoma may also develop through progression from diffuse astrocytoma (WHO grade II) or anaplastic astrocytoma (WHO grade III); and these are termed “secondary glioblastomas”. They are much less frequent than primary glioblastomas (<10% of all glioblastomas), and typically develop in younger patients (mean, 45 years) (Figure 1.4). The time to progression from diffuse astrocytoma WHO grade II to glioblastoma varies considerably, with time intervals ranging from less than 1 year to >10 years, the mean interval being 4– 5 years. Survival of patients with secondary glioblastoma is significantly longer (median survival time, 7.8 months) than for those with primary glioblastoma (4.7 months), but this is likely to be the reflection of younger age of secondary glioblastoma patients [21,39].
36 Astrocytic tumours
62 years) {1620, 1625A}. Neuroimages of the dynamic growth of a primary glioblastoma and a secondary glioblas-toma are shown in Fig. 1.30. Glio-blastoma may also develop through progression from diffuse astrocytoma (WHO grade II) or anaplastic astro-cytoma (WHO grade III); and these are termed “secondary glioblastomas”. They are much less frequent than primary glioblastomas (<10% of all glioblastomas) {486, 1620, 1625A}, and typically develop in younger patients (mean, 45 years). The time to progression from diffuse astrocytoma WHO grade II to
glio-blastoma varies considerably, with time intervals ranging from less than 1 year to >10 years {1627}, the mean interval being 4–5 years {1625, 2371}. Survival of patients with secondary glioblastoma is significantly longer (median survival time, 7.8 months) than for those with primary glioblastoma (4.7 months), but this is likely to be the reflection of younger age of secondary glioblastoma patients {1620, 1625}. Primary and secondary glioblastoma constitute relatively distinct disease entities that evolve primarily through different genetic pathways, show different expression
profiles {1625}, and are likely to differ in response to therapy, but share a high frequency of LOH 10q, which is likely to be associated with the overall glio-blastoma phenotype {620, 622, 1620, 1625A}.
Histopathology
Glioblastoma is an anaplastic, cellular glioma composed of poorly differentiated, often pleomorphic astrocytic tumour cells with marked nuclear atypia and brisk mitotic activity. Prominent microvascular proliferation and/or necrosis are essential diagnostic features.
As the term glioblastoma “multiforme” suggests, the histopathology of this tumour is extremely variable. While some lesions show a high degree of cellular and nuclear polymorphism with numerous multinucleated giant cells, others are highly cellular, but rather monotonous. The astrocytic nature of the neoplasms may be easily identifiable, at least focally, in some tumours, but difficult to recognize in others owing to the high degree of anaplasia. The regional heterogeneity of glioblastoma is remarkable and poses challenges to histopathological diagnosis on specimens obtained by stereotaxic needle biopsies {254}.
Tissue patterns
The diagnosis of glioblastoma is typically based on the tissue pattern rather than on the identification of certain cell types. The presence of highly anaplastic glial cells, mitotic activity and vascular proliferation and/or necrosis is required. The distribution of these key elements within the tumour is variable, but large necrotic areas usually occupy the tumour centre, while viable tumour cells tend to accumulate in the periphery. The circum-ferential region of high cellularity and abnormal vessels corresponds to the contrast-enhancing ring seen radio-logically. Vascular proliferation is seen throughout the lesion, often around necrotic foci and in the peripheral zone of infiltration.
Secondary structures
The migratory capacity of glioblastoma cells within the CNS becomes readily apparent when they reach borders that constitute a barrier: tumour cells line up and accumulate in the subpial zone of the cortex, in the subependymal region, around neurons (“satellitosis”), and about Fig. 1.30 Rapid evolution of a primary glioblastoma in a 79 year-old patient (upper). MRI shows a small cortical lesion
that within 68 days developed into a full-blown glioblastoma with perifocal edema and central necrosis. This is in contrast to the development of secondary glioblastoma through progression from low-grade astrocytoma (lower).
Figure 1.4 Neuroimages of the dynamic growth of a primary glioblastoma and a secondary glioblastoma. Rapid evolution of a primary glioblastoma in a 79 year-‐ old patient (upper). MRI shows a small cortical lesion that within 68 days developed into a full-‐blown glioblastoma with perifocal edema and central necrosis. This is in contrast to the development of secondary glioblastoma through progression from low-‐grade astrocytoma (lower)[19].
Primary and secondary glioblastoma constitute relatively distinct disease entities that evolve primarily through different genetic pathways, show different expression profiles, and are likely to differ in response to therapy, but share a high frequency of LOH 10q, which is likely to be associated with the overall glioblastoma phenotype (Figures 1.5 and 1.6) [23,40].
