7

2.0 REVIEW OF LITERATURE

A number of diseases such as diarrhea, paralysis, diabetes, tumor etc affect the lifespan of an individual; they may be due to environmental, genetic or pathogenic factors. Tumor is such a disease in which normal growth control mechanism fails, uncontrolled cell proliferations produces a growing mass of cell called tumor or neoplasm. Tumors are classified as benign tumor and malignant tumor based on their likelihood of spreading (Greenlee et al 2000). According to David et al 2007, the fourth edition of the World Health Organization (WHO) classification of tumor of the Central Nervous System published in 2007, lists several new entities (Table 2.1). This classification was initiated through a resolution of WHO executive board in 1956 and the World Health Assembly in 1957. Its objectives have remained the same till date to establish a classification and grading of human tumors that is accepted and used worldwide. Without clearly defined histopathological and clinical diagnostic criteria, epidemiological studied and clinical trial could not be conducted beyond institutional and national boundaries (Greenlee et al 2000). The 2007 WHO classification of tumor of Central Nervous System are as follows:

Table No. (2.1) Classification of Central Nervous System (Greenlee et al 2000)

Tumor type Tumor of neuroepithelial tissue

Subtype Astrocytic tumor, oligodendroglial tumors, oligoastrocytic tumors, ependymal tumors, choroid plexus tumors, tumor of pineal regions embroynal tumors, neuronal and mixed neuronal glial tumors Schwannoma, neurofibroma, perinurloma, malignant peripheral Tumor of meningothelial cells, mesenchymal tumors, primary melanocytic lesions and other neoplasm related to the meanings Malignant lymphomas, plastocytoma, granulotic sarcoma Germinoma, teratoma, embroynal carcinoma Granual cell tumor Carcinoma

Tumor of cranial and paraspinal nerves Tumor of meanings Lymphomas and hematopoietic neoplasm Germ cell tumors Tumor of sellar regions Metastataic tumor

Primary brain tumors are commonly located in the posterior cranial fossa in children and in the anterior two thirds of the cerebral hemisphere in adults, although they can affect any part of the brain (Greenlee et al 2000). In the United State in the year 2005 it was estimated that there were 43,8000 new cases of brain tumor (Central Brain Tumor Registry of the United state, primary brain tumor in the United States, statistical report 20052006) (Greenlee et al 2009) which accounted for 1.4% of all cancers, 2-4% of all cancer deaths and 20-25% of pediatric cancer. Ultimately, it is estimated

that there are 1, 30, 000 deaths/year in the United States alone as a result of brain tumors (Chamberlain et al 2008). In the US, approximately 2000 children and adolescents younger than 20 years of age are diagnosed with malignant brain tumor each year. Higher incidence rates were reported in years 1985-94 than in years 1975-84 (Gurney et al 2008). One theory proved that it is the result of improved diagnosis and reporting since the jump occurred at the same time as MRIs became available widely and since there was no coincident jump in mortality. The CNS cancer survival rate in children is approximately 60%. The rate varies with the age of onset, with younger patients having mortality and cancer type have high rate than children (Gurney et al 2008). In children under age 2 years about 70% of brain tumors are medulloblastoma, ependymoma and low grade glioma (figure 2.1). But in infants teratoma and atypical terotoid rhabdoid tumor (Jeanne et al 2000) are found. Germ cell tumors including teratoma make up just 3% of pediatric primary brain tumors but the worldwide incidence varies significantly (Echevarria et al 2008).

Figure (2.1): The brain stem glioma in 2 year old children

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Symptoms of brain tumor may depend on two factors: tumor size (volume) and tumor locations. The time of symptoms and disease correlates in many cases with the nature of tumor (benign or malignant). It is a frequent reason for seeking medical attenuation in brain tumor cases. Large tumor or tumors with extensive perifocal swelling edema inevitably lead to elevated intracranial headaches, pressure (hypertension), vomiting (sometimes which translates nausea), clinically state into of without altered

consciousness (Coma), dilatation of the pupil on the side of the lesion (anisocoria), however even small tumor obstructing the passage of cerebrospinal fluid (CSF) may cause early signs of increased intracranial pressure (which result in displacement of certain parts of the brain such as cerebellar tonsils or the temporal uncees resulting in lethal brain stem compression). Elevated intracranial pressure in young children may cause an increase in the diameter of the skull and bulging of the fontanelles (Stark et al 2003). Depending on the tumor location and damage it effect the surrounding brain structures, either through compression or infiltration, any type of focal neurologic symptoms may occur such as cognitive and behavioral impairment, personality changes, hemiparesis, hyperthesia, aphasia ataxia, visual field impairment, facial paralysis, double vision tremor etc. (Amariglio et al 2009). Gliomas are non metastatic CNS neoplasm that arises from glial cells (e.g. astrocytes and oligodendrocytes) but the actual cell of origin for gliomas is unknown. In adults, astrocytic tumors are classified by the world health

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organization as grade I (pilocytic astrocytoma), grade II (oligoastrocytoma), grade III (anaplastic astrocytoma) [AA] and grade IV (Glioblastoma Multiforme) [GBM]. The high grade or malignant astrocytomas (AAs and GBM) comprise the most common type of primary CNS tumor with a combined incidence of approximately 3/100,000 person/years. GBM is the most commonly diagnosed primary malignant brain tumor in adults with an incidence of 2-6/100,000 person/years (Surawicz et al 1999). The median survival time for newly diagnosed GBM patients treated with surgery, radiation therapy and chemotherapy is only approximately 11-12 months (Simpson et al 2003), only 3% patients survive for 5 years (Davis et al 1999). The medial survival time for anaplastic astrocytoma patients is approximately 22 months with a 31% free survival time for 6 months progression (Wong et al 1999). In this review, the term gliomas which included nonastrocytic tumor such as oligodendrogliomas or specifically to astrocytomas (Galanis et al 2006). According to WHO, gliomas are the tumor of neuroepithelial tissue. In adult, neuroepithelial tumors are classified as follows (figure 2.2) (Greenlee et al 2000):

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Figure (2.2): Tumor of neuroepithelial tissue

Astrocytic Tumor

Oligodendroglial Tumor

Ependymoma Tumor

Choroid Plexus Tumor

Low grade tumor

High grade tumor

Grade I

Grade II

Grade III

Grade IV

Pilocytic Astrocytoma

Oligoastrocytic Subependymoma

Anaplastic Tumor

Glioblastoma Multiforme

Malignant gliomas may manifest at any age including congenital and childhood cases. Peak incidence is however in adults older than 40 years and adult males are more frequently affected than females (Preusser et al 2006). Invasions of glioma cells requires interaction with the extra cellular matrix and with surrounding cell of the healthy brain tissue such as vascular proliferates and tissue necrosis are characteristic features of malignant gliomas in particular glioblastoma multiforme. These features are most likely the consequence of rapidly increasing tumor mass that inadequately oxygenized by the pre-existing vasculature. In malignant glioma distinct

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molecular pathways including the P53 pathway shows frequent alterations that seem to be pathogenetically relevant (Preusser et al 2006). The role of neuropathology and neurobiology provided classifications of brain tumor to clarify etiology, pathogenesis of brain tumor as rational basis for development of new diagnostic tests, therapies and to translate testing for new clinically relevant molecular parameters into clinical applications (Preusser et al 2006). Pediatric denovo/primary glioma are rare than adult gliomas. Relatively few studies have been performed on molecular properties of pediatric gliomas. The low grade gliomas that occur in childhood are different from adult gliomas. They are more likely to involve the cerebellum, midbrain, optic pathway, optic nerve, optic nerve crossing, posterior optic pathway, dysembryoplastic neuroepithelial tumor and desmoplastic cerebral astrocytoma. The type and grade of gliomas are also different as well as the rate of survival of patients (Burger et al 2002). Gliomas can be diagnosed by slowly progressive focal neurological signs and signs of elevated intracranial pressure as well as epilepsy in a patient with a negative history. However a sudden onset of symptoms such as an epileptic seizure in a patient with no prior history of epilepsy, sudden intracranial hypertension (this may be due to bleeding within the tumor, brain swelling or obstruction of cerebrospinal fluids passage) is also possible (Mischel et al 2003). Abundant evidence suggests the presence of unrecognized, clinically relevant subclasses of the diffuse gliomas, both with respect to their

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underlying molecular phenotype and their clinical response to therapy (Mischel et al 2003). It is also apparent from clinical response to a variety of treatment that histologically identical tumors can behave in highly different manners such as posterior fossa, pineal regions and so on (Mischel et al 2003). This can result in underpowered studies that fail to detect potentially promising new therapies (Betensky et al 2002). For instance, a small subset of patients can have a substantial response to irinotecan, whereas the majority of patients do not respond for the same (Cloughesy et al 2003). These findings underscore the fact that histopathological evaluation may not be sensitive to much of the variation in underlying biology. As oncologists move toward molecularly targeted therapies, identification of distinct molecularly defined subgroups becomes critical (Mischel et al 2003). To additionally clarify the underlying biology and to refine survival prognostication, adjunctive studies of tumor-associated genes such as the epidermal growth factor receptor (EGFR), tumor protein p53 (TP53), phosphatase and tensin homologue (PTEN), proliferation related Ki-67 antigen (Ki-67), murine double minute 2 (MDM2), mut-s homologue 2 (MSH 2), cyclin-dependent kinase inhibitor 2A (CDKN2A), sarcoma amplified sequence (SAS) and platelet derived growth factor (PDGFA) have been used (Tortosa et al 2001, Bouvier et al 1998, Stark et al 2003, Li et al 1997). In general, individual gene or protein assays alone or in combination with histologic features are neither predictive of survival of gliomas patients nor able to guide therapeutic decisions. Thus, although these genes may indeed play a role in gliomas biology, their utility as diagnostic markers is not yet clear, perhaps due to heterogeneity within the tumor groupings (Mischel et al 2003).

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Microarray analysis offers the option of quantitative and reproducible tumor evaluation by simultaneously evaluating thousands of individual gene expression measurements. This approach has been applied to many different cancers including gliomas (Mischel et al 2003, Kim et al 2002, Ljubimova et al 2001, Nutt et al 2003, Rickman et al 2001, Sallinen et al 2000, Shai et al 2003, Tanwar et al 2002). Unlike pathological evaluation, microarray analysis provides the means to describe the underlying variation in genetic composition of the tumors allowing for novel tumor class identification and patient prognostication. Efforts at dissecting gliomas into more homogeneous groups with clear relationships to underlying molecular defects using microarrays have suggested that different types of gliomas have different gene expression profiles (Shai et al 2003) and there is evidence for distinct molecular subsets of morphologically identical gliomas (Mischel et al 2003) and gene expression based predictor more correlates with survival than with histological type (Nutt et al 2003). Glioblastoma multiforme and anaplastic astrocytoma have been associated with the genetic acute hepatic porphyries including positive testing associated with drug refractory seizures. Unexplained complications associated with drug treatment with these tumors should alert physician to as undiagnosed neurological porphyria (Nutt et al 2003). Imaging plays a central role in the diagnosis of brain tumors. Early imaging methods such as pneumoencephalography and cerebral angiography have been abandoned in recent times in favour of non invasive, high

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resolution modalities such as computed topograph and especially Magnetic Resonance Imaging. Besides these methods the definitive diagnosis of brain tumor can only be confirmed by histological examination of tumor tissue sample obtained either by means of brain biopsy or open surgery. The histological examination is essential for determining the appropriate treatment and the correct prognosis. This examination performed by a pathologist, typically has three stages such as interpretative examination of fresh tissue, preliminary microscopic examination of prepared tissue and follows up examination of prepared tissue after immunohistochemical staining or genetic analysis (Amariglio et al 2009). Immunohisochemistry (IHC) has emerged as a powerful investigative tool that can provide supplemental information to the routine morphological assessment of tissues. The use of IHC to study cellular markers that define specific phenotype has provided important diagnostic, prognostic and predictive information relative to disease status and biology. The application of antibodies to the molecular study of tissue pathology has required adaptation and refinement of immunohistochemical technique particular for use in fixed tissues (Frederick et al 2000). Many antibodies such as S100, nestin, glial fibrillary acidic protein (GFAP), neuron specific enolase (NSE), epidermal growth factor receptor (EGFR), vascular endothelia growth factor (VEGF), desmin, vimentin, matrix metalloprokinase 2 (MMP 2), matrix metalloproteinase 9 (MMP 9) and any other are used to diagnosed the different type of brain tumor depending on their binding site. To evaluate the

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alteration in pediatric gliomas with special reference to protein expression, we examined the expression of epidermal growth factor receptor in different grade of gliomas. The epidermal growth factor receptor (EGFR, ErbB-1 and HER1 in human) is the cell surface receptor for member of the epidermal growth factor family (EGF Family) of extra cellular protein ligands (Kim et al 2001). The epidermal growth factor receptor is a member of the ErbB family of receptor of the subfamily of four closely related receptor tyrosine kinase such as EGFR (ErbB-1), HER 2/e-new (ErbB-2), HER 3 (ErbB-3) and HER 4 (ErbB4). Mutation affecting EGFR expression or activity could result in cancer (Zhang et al 2007). Upon activation by its growth factor ligand, EGFR undergoes transition from an inactive monomeric form to an active homodimeric form as shown in figure (2.3), although there is some evidence that performed inactive dimers may also exist before ligand binding. In addition to forming homodimer after ligand binding EGFR may pair with another member of the ErbB-2/HER 2/neu, to create an activated heterodimer (Boonstra et al 2010).

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Figure (2.3): Photomicrographs of a tumor without EGFR overexpression and a positive sample with strong membrane staining for EGFR

Mutation that leads to EGFR over expression knows as upregulations or over activity has been associated with a number of cancers, including lung cancer and glioblastoma multiforme. In this latter case a more or less specific mutation of EGFR, called EGFRvIII is often observed (Kuan et al 2001). Mutation involving EGFR could lead to its constant activation which could result in uncontrolled cell division (Lynch et al 2004). Consequently mutation of EGFR has been identified in several types of cancer and it is the target of an expanding class of anticancer therapies (Zhang et al 2007). The identification of EGFR as an oncogene had led to the development of anticancer therapeutics directed against EGFR, including gefitinib and erlotinib for lung cancer and cetuximab for colon cancer (Paez et al 2004). In the cancerous precursor cell, if amplification or overexpression of EGFR occur then it mutated the chromosome number 10 which cause primary glioblastoma and if the mutation occurs in platelet derived growth factor it altered the retinoblastoma gene which cause mutation and cause secondary glioblastoma. Primary glioblastoma and secondary glioblastoma combinedly results in glioblastoma multiforme (figure 2.4) (Paez et al 2004).

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Figure (2.4): Genetic pathways leading to primary and secondary gliomas. (EGFR = epidermal growth factor receptor, PTEN = phosphatase tensin
homology gene on chromosome 10, PDGF = platelet-derived growth factor, FGF2 = fibroblast growth factor 2, Rb = retinoblastoma gene, CDK4 = cyclin-dependent kinase 4, GBM = Glioblastoma Multiforme). MMAC = mutated in multiple advanced cancers, DCC = deleted in colorectal carcinoma, LOH = loss of heterozygosity).

Many therapeutic approaches are aimed at the EGFR, Cetuximab and panitumumab are examples of monoclonal antibody inhibitors. However the EGFR is of the IgG1 type and the latter is of the IgG2 type, consequences on antibody dependent cellular cytotoxicity can be quite different (Yan et al 2005). Other monoclonal antibodies in clinical development are Zalutumumab, nimotuzumab, matuzumab, gefitinib, erlotinib and lapatinib (the latter still in clinical trial) are examples of small molecular kinase inhibitors. The monoclonal antibodies block the extracellular ligand domain

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with the binding site blocked; signal molecules can no longer attach these and activate the tyrosine kinase. Another method is using small molecule to inhibit the EGFR tyrosine kinase, which is on the cytoplasm side of the receptor. Without kinase activity EGFR is unable to activate itself, which is a prerequisite for binding of downstream adaptor proteins. Obstensibly by halting the signaling cascade in the cells that rely on this pathway for growth, tumor proliferation and migration is diminished (Adams et al 2005). EGFR-1 is a humanized monoclonal antibody (MAb) (IgG1 isotope) that recognized an epitope located in the extra cellular domains of EGFR with the binding site blocked, signal molecule can no longer attach there and activate the tyrosine kinase. EGFR has been studied extensively as a target for direct immune attack via specific antibodies like EGFR-1 (Mateo et al 1997). The trial was designed to access safety and immunogenicity of EGFR-1 in high grade pediatric gliomas. In summary, EGFR-1 is a good sensitive marker for gliomas prognosis (Tania et al 2006). Over expression of epidermal growth factor receptor (EGFR, EbrB-1) correlates with enhanced malignant potential of many human tumor types including glioblastoma multiforme. The significance of EGFR expression in meningiomas is however unclear. Reports regarding the other EGFR family members, ErbB-2 and ErbB-4 in brain tumor are rare. In this study the expression of the EGFR family members was analyzed in relation to various parameters for the clinical importance of these receptors in 44 gliomas and 26 meningiomas. In gliomas quantitative real time reverse transcription (RT) PCR revealed the highest EGFR mRNA expression in high grade gliomas. In

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contrast, ErbB-4 expression was most pronounced in low grade gliomas. Immunohistochemistry showed significantly higher EGFR protein expression in high grade gliomas compared to low grade gliomas (p = 0.004). ErbB-2 protein expression was mainly seen in high grade gliomas, ErbB-3 protein expression was low in all gliomas analyzed, ErbB-4 protein expression was significantly higher in low grade gliomas (p = 0.007). In meningiomas, quantitative real time PCR revealed expression of EGFR, ErbB-2 and ErbB-4 mRNA in the majority of the tumor. ErbB-3 was detected in only one of the meningiomas analyzed. Immunohistochemistry demonstrated higher ErbB-2 protein expression in meningiomas. An intriguing observation in astrocytomas and oligodendrogliomas grade II was a significantly decreased overall survival for patients with high EGFR protein expression (p= 0.04) (Anderson et al 2004). Oligodendrogliomas are tumor composed of cells that histologically resemble the mature oligodendrocytes, occurring predominately in the cerebral hemispheres. Constituting approximately 5% of all intracranial CNS tumor, oligodendrogliomas are most commonly diagnosed during the fourth and fifth decades of life with many patient having a much year history of epileptic seizure activity that suggests a prolonged prediagnostic period with tumor (Chin et al 1980) even in patients with such a prolonged history, postoperative survival are disproportionately short. However progress in the therapy of these tumors is being made (Cairncorss et al 1994). Recent work on oligodendrogliomas has revealed that they typically respond initially to the chemotherapeutic protocols that depend on alkylating chemotherapeutic agents such as PCV (Levin et al 1980).

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At present, the mean post diagnostic survival for low grade oligodendrogliomas is 10 years, although for high grade tumor the survival is only 5 years (Paleologos et al 1999). Other methods of therapy are needed if post operative survivals in high grade tumor are to be extended (Paleologos et al 1999). EGFRwt and EGFRvIII also represent target in astrocytic neoplasm because a high percentage of astrocytic tumor express either antigen or both. Although EGFRwt is present on the benign surface of leptomeninges, no study to date has identified the antigen within the CNS parenchyma. Up to 90% of high grade astrocytic gliomas express the EGFRwt antigen, a hyper expression associated with EGFR gene amplification is greater than 50% of glioblastomas. Previous studied indicate that oligodendrogliomas also express the antigen, although genomic amplifications is generally absent (Broholm et al 1999). Approximately 50% of high grade astrocytomas contain the EGFRvIII rearrangement that result in a 145 KD molecule of a unique primary sequence characterized by an inserted glycine at the position of frame deleted amino acid 5 and 274. EGFRvIII would be a very attractive target if identified in oligodendrological tumor. To date one study has identified that one oligodendroglial tumor with genomic rearrangement (Reifenberger et al 1996). Molecular as well as immunohistochemical study has not been studied in oligodendroglial tumor till date. On the basis of previous work on astrocytic gliomas, EGFRwt and EGFRvIII if expressed in oligodendroglial tumor may offer attractive target for immunotherapeutic approaches to these tumors. The EGFR & EGFRvIII

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protein distributions and intensity among 50 low and high grade oligodendroglial tumors and 10 GBM exhibiting a significant population of cells with oligodendroglial features (figure 2.5). Furthermore the study to characteristic EGFRwt and EGFRvIII distribution and heterogeneity in the vascular and parenchymal components among the grades of these tumors (Rogar et al 2000).

Figure (2.5): EGFR protein distribution in oligodendroglioma

Ependymomas account for 2-9% of all neuroepithelial tumors, amounting to 6-12% of all intracranial tumors in children and up to 30% of those in children younger than 3 years. Recent finding provided evidence that intracranial spinal ependymomas share similar molecular profiles with the radial glial cells at their corresponding locations (Rshunov et al 2000). Although an anaplastic variant is recognized, numerous studies examining the prognostic implication of histological features such as necrosis, endothelial proliferation and mitosis have yielded contradictory results. In order to improve outcome prediction in affected patients and to refine therapeutic decision making, there is a strong need for identifying relevant biological correlates of tumor behavior. The molecular biology of tumors is a rapidly, expanding field and include investigation into cytogenetic oncogenes, growth

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factors, growth factor receptors, hormonal receptors, proliferation markers, apoptosis, cell cycle genes and cell adhesion molecules as well as factor potentially related to therapeutic resistance such as the multi-drug resistance gene. The molecular biology of astrocytoma tumor in adults has been the subject of many studies however relatively few studies have been forced on ependymomas oncological markers in ependymomas that have been identified to date and highlight the limitation as a brain for defining area for further investigation (Ronald et al 2008). Prognostic significance based on immunohistochemistry of ependymomas has been reported; in few studies 80 patients with intracranial ependymomas were examined retrospectively for immunoexpression of various tumors associated antigen and apoptosis. The result demonstrated significant preponderance of depression of the tendency of vascular endothelial growth factor (VEGF), epidermal growth factor receptor (EGFR) and P53 protein in high grade tumors (Korshunav et al 2000). Epidermal growth factor receptor (EGFR) and transferrin receptor (TfR) level were determined in 14 intracranial neoplasms (4 glioblastoma multiforme, 4 meduloblastomas, 4 ependymomas, one cerebellar astrocytoma and one anaplastic astrocytoma) and in 4 samples of normal brain tissue. A competitive radio receptor assay with 1251 EGFR and 1251 transferrin was performed using the primitive neuroectodermal tumor derived TE-671 tissue culture cell line as a standard. EGFR were present on TE-671 cells of all 4 ependymomas and 2 of the 4 GBM tissues. The number of EGFRs per cell for ependymomas were estimated to range from 1000 to 6000. Transferrin

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receptors were detected on TE 671 cells, 2 of 4 medulloblastoma and 1 of the 4 GBM tissues (Hall et al 2008). A cell surface binding assay was performed and analyzed directly on the rat ependymomal cell monolayer. The identification of EGFR on 3 ependymomas and TfR on the meduloblastomas suggests that malignant central nervous system tumors that spread by cerebrospinal fluid pathway may be treatable by intrathecal antibody toxin conjugates. The presence of EGFRs on ependymomas may reflect a role of the receptor in the malignant phenotype of this tumor (Hall et al 2008).

Figure (2.6): EGFR expression in ependymomas

Astrocytic tumors are the most common tumor of the central nervous system. The mechanism of genetic change of astrocytic tumor has not been understood completely. Recently, EGFR and survivin has been identified as a member of the inhibitor of apoptosis family. EGFR expression is considered as important prognostic factor of many tumors (Yoshinori et al 2003). Epidermal growth factor receptor (EGFR) is commonly over expressed in adult high grade gliomas such as in astrocytoma grade III and glioblastoma multiforme, 40-50% of tumors demonstrated amplification of the EGFR gene

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often with rearrangement and constitutive activation of the gene product suggesting that EGFR might play a role in the malignant progression of these neoplasms. In this regard several groups have shown that over expression of EGFR is associated with an adverse outcome in adult gliomas. In contrast to the extensive studies of EGFR that have been performed in adult astrocytoma, little information has been reported about EGFR over expression and amplification as well as their prognostic relevance in pediatric astrocytoma, which carry a somewhat more favorable prognosis than their adult counterparts. To address this issue, the expression of EGFR using immunohistochemistry and screening for amplification of the EGFR gene using a competitive PCR in a series of 27 archival pediatric astrocytoma treated consecutively (Bredel et al 1999). Tumors were categorized based on protein expression patterns and the association between expression statuses. Although elevated immunoreactivity for EGFR was observed in 80% of tumors, only 2 of the cases had gene amplification. No difference in outcome was observed between tumors that exhibited extensive EGFR immunoreactivity and those that did not (p>0.3). Although EGFR expression did not seem to be of prognostic relevance for the outcome of pediatric patients harboring astrocytoma, consistently high level expression of EGFR in these neoplasm suggest that this receptor plays a role in the malignant phenotype of these tumors as shown in figure (2.7). Accordingly, treatment approaches targeting EGFR might be of potential therapeutic benefit for astrocytoma of children (Bredel et al 1999).

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Figure (2.7) EGFR expression in astrocytoma

As shown in figure (2.8) glioblastoma are the most common malignant brain tumors in adults and they are notoriously difficult to treat successfully. The glioblastoma is a very serious and challenging disease and the survival is usually a year on average (Natalie et al 2005). Based on results from other studies, the researchers hypothesized that variations in several different genes might play a role in the tumors response to EGFR inhibitors. They looked for mutations in genes called EGFR and HER 2/c-neu and they analyzed the activity of EGFR, EGFR variant (EGFRvIII) and a gene called PTEN. Many tumors, other than brain tumors have mutations or abnormal activity of one or more of these genes, which help to control cell growth and other functions (Natalie et al 2005).

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Fig (2.8): Cross appearance of glioblastoma multiforme

Glioblastoma that produced both EGFRvIII and PTEN were 51 times more likely to shrink when treated with EGFR inhibitors than tumors without this combination of proteins, the researchers found patients whose tumors expressed these proteins and who received an EGFR inhibitor went almost 5 times longer on average before their tumors progressed (243 days versus 50 days) than those whose tumors did not express both of the proteins. In contrast, EGFR and HER-2/c-neu activity had no effect on how tumors responded to these drugs. Similar results were found in tissues from another group of 33 glioblastoma patients who had taken part in a clinical trial of erlotinib drug at the University of California, San Francisco (Natalie et al 2005). The finding suggested that both EGFRvIII and PTEN proteins are important for tumors to be susceptible for EGFR inhibitors. According to Mischel, EGFRvIII may act to sensitize glioblastoma cells, while PTEN loss may act as a resistance factor. The researchers tested their results in several different cell models and repeatedly found that expression of these two proteins made the cells sensitive to EGFR inhibitors and that PTEN less promoted resistance in those models (Natalie et al 2005).

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The study also revealed important information about how glioblastoma and other tumors develop. According to Mischel, EGFRvIII and PTEN play critical roles in tumor response to treatment could lead to new combination therapies that target both proteins. Such therapies might also be beneficial for other types of cancer (Natalie et al 2005). Over the years, our knowledge of GBM biology has steadily improved. From a molecular standpoint, GBMs are highly heterogeneous tumor with multiple signaling pathways differently activated or silenced with converging and parallel complex interactions (Amariglio et al 2009). It is these intricacies that are thought to confer GBM with its notorious plasticity in response to therapeutic interventions. Therefore, a major challenge in the clinic is to determine the appropriate events to target. The most common genetic abnormality in GBMs is the activation of receptor tyrosine kinases (RTKs), of which aberrant expression of EGFR is the most frequent (Amariglio et al 2009). Concomitant with EGFR gene amplification event is the occurrence of an intragene in frame deletion of exons 2-7 of the EGFR gene. This rearrangement product known as EGFRvIII, codes for a ligand independent receptor, which is constitutively activated and highly oncogenic (Pedersen et al 2001). During EGFR locus amplification, a portion of the amplicon rearranges to produce EGFRvIII, leading to the co-expression of EGFRwt and EGFRvIII within the same cells. Alternatively, if the rearrangement occurs early during the amplification process, most cells will express the EGFRvIII variant predominantly with very little. Thus, in individual human GBMs,

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expression of EGFRWT, EGFRWT/vIII or EGFRvIII is observed (Ekstrand et al 2010, Wong et al 1999, Biernat 2004). Although the prognostic value of EGFRvIII expression is still debated, recent molecular characterization of targeted therapy resistance appear to indicate that EGFRvIII confers properties distinct from EGFRwt (Mellinghoff et al 2007). To understand EGFR signaling in GBM to better predict efficacy of targeted therapeutics, researchers developed 3 preclinical models of GBM based on over expression of EGFRwt alone, co expression of EGFRwt and EGFRvIII and expression of EGFRvIII alone, thus reflecting natural occurrences in human GBMs. Using these models, researchers showed ectopic expression of EGFR (both wt and vIII) in adult CNS tissues. In the context of p16Ink4a/p19ARF and PTEN inactivation, it leads to the formation of GBMs de novo. Researchers also show that EGFR-mediated tumor formation is accompanied by the activation of canonical and unexpected signaling pathways. Their findings showed that the study of EGFR expression in this type of tumor play the genetic contributors to gliomagenesis (figure 2.9) and therapeutic treatment resistance in GBMs (Mellinghoff et al 2007).

Figure (2.9): EGFR expression in glioblastoma multiforme

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The study of the presence of the epidermal growth factor receptor (EGFR), interleukin-13 receptor (IL-13R), interleukin-4 receptor (IL-4R) and transferrin receptor (TfR) determined by immunoflourescence microscopy, amount in freshly frozen tumor samples from 38 patients with high grade gliomas (glioblastoma multiforme or anaplastic astrocytoma). The frequency of high receptor expression was 32 of 38 (84%) for EGFR, 30 of 38 (79%) for IL-13R, 25 of 38 (66%) for TfR and 17 of 38 (45%) for IL-4R reactivity. Normal brain endothelium was observed for TfR and reactivity of normal brain astrocytes was observed for IL-4R. Because of cross-reactivity of interleukin-13 with the IL-4R receptor, researchers analyzed the reactivity of interleukin-13 with normal astrocytes. In contrast, EGFR expression was not observed in normal brain. A number of patients (10 of 38 patients) showed unequal expression of EGFR and IL-13R. Thus, some patients may benefit more from interstitial therapy with an EGFR and EGFR directed fusion protein than from therapy with IL-13R directed fusion protein and vice versa. The safety profile may be improved with an agent directed to EGFR versus agents directed to TfR, IL-4R or IL-13R. Design of clinical trials of fusion proteins in patients with brain tumors may be enhanced by inclusion of relevant receptor density measurements (Stephen et al 2003). As a result patients with malignant gliomas do not respond to any current therapy. Epidermal growth factor receptor (EGFR) controls several oncogenic processes being frequently up-regulated in gliomas due to overexpression, gene amplification and gene mutation. EGFR inhibitors are being tried in gliomas, yet the molecular determinants of the therapeutic response are unclear. EGFR amplification was determined by

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immunohistochemically and chromogenic in situ hybridization (CISH) in 27 primary glioblastomas, 24 anaplastic oligodendroglioma (AO) and 4 anaplastic oligoastrocytoma (AOA). EGFR over expression was associated with EGFR amplification, being found in 48-53% in GBM, 33-40% in anaplastic oligodendroma and 67-75% in anaplastic oligoastroytoma, respectively. EGFRvIII was found in 22% GBM, 8% anaplastic oligodendroma and was absent in anaplastic oligoastroytoma. No association was observed between EGFR alterations and patient survival. Thus researchers characterized EGFR molecular alteration in Portuguese patient with malignant glioma and identified a subpopulation of patients presenting putative biomarker for EGFR based therapies (Viana et al 2008). Thus after these above studies it is characterized that, there are very few non-profit organizations that are dedicated to promoting more productive and aggressive research into the cause(s) of eventual cure for glioblastoma multiforme. To our knowledge, there are no organizations that are solely dedicated to assisting families with their financial needs as they are dealing with a loved one that is suffering from this horrific form of cancer. Parents of younger GBM patients must take time off from work and sadly nearly 100% of all GBM patients will die within a relatively short period of time.