Glioblastomas: Molecular Diagnosis and Pathology

Abstract

Glioblastoma (GBM) is a fatal human brain tumor of grade IV/4 by WHO classification, with a very poor prognosis. At the molecular level and clinical, GBM has at least two types, primary and secondary. Each has a different tumorigenesis and clinical presentation. In this chapter, some major molecular biomarkers and diagnostic hallmarks of GBM will be reviewed and discussed.

1. Introduction

Glial tissue in the human brain includes astrocytes, oligodendrocytes, microglia, and ependymal cells, and each cell type has its own function. Like oligodendrocytes, proving the myelin sheath covering the axons, making the signal transporting faster. While the ependymal cells cover the surface of ventricles. When a specific mutation happens, each glial cell may produce its own glioma (glial neoplasm), the terminology of the glioma will follow the origin of the glial cells. Like oligodendrocyte-original glioma named as oligodendroglioma. Each glioma has different grading, which indicates the tumors’ malignancy as well as the clinical behavior. Such as adult’s astrocytomas have three grades, from grade 2 to grade 4, the highest grade, (CNS WHO grade 4) astrocytoma also called glioblastoma (GBM). GBM is the most common malignant brain tumor and accounts for 46% of primary malignant brain tumors, which occurs in older patients with a mean age of 64 years old. The most common location of GBM is in the supratentorial region (frontal, temporal, parietal, and occipital lobes), with the highest incidence in frontal lobe, rarely occurs in the cerebellum and spinal cord. GBMs show on MRI scan an enhancing lesion, after administration of contract agent, heterogenic enhancing, or ring-enhancing mass lesion will be presented (Figure 1) GBM is a malignant neoplasm, by current treatment including surgery, chemo, and radiation therapy, most patients with GBM have only about 15 months of survival time due to the aggressive nature of this tumor and some other reasons.

Figure 1.

On this patient, the left parietal heterogenic enhancing mass lesion is most likely a GBM (MRI scan, A. coronal, B. sagittal, and C. axial). An autopsy gross picture of a GBM on the right hemisphere with focal invasion into corpus callosum (D).

Annual age-adjusted incidence rates for GBM have increased in recent years to 3–6 cases per 100,000 people, as the reports from the USA, Canada, UK, and Australia [1].

In the last two decades, the research discovered that GBMs have two different subtypes by their distinct genetic alteration, each subtype has its own clinical behavior and molecular background. This review will briefly cover this knowledge of the current understanding of GBMs, and include some diagnostic and brief molecular information about this malignant brain neoplasm.

1.1 Migration and metastasis of GBM

Biologically, astrocytomas, no matter low or high grade, are characterized by their infiltrating growth. For example, GBM usually deeply infiltrates the white matter of the brain and sometimes goes to cross the corpus callosum and makes a terrible butterfly pattern in MRI scan (Figure 2A). This nature of infiltrating growth makes the astrocytoma one of the most challenging tumors for surgical resection, since no distinctive clear surgical margin can be archived without damaging the brain function during the tumor resection surgery by neurosurgeons.

Figure 2.

A 52-year-old male with newly diagnosed GBM showed a butterfly sign on MRI scan (A). (see yellow arrows). A patient with right temporal GBM (B) and surgically resected successfully, but years later another nodule with the feature of GBM (C) in the left cerebellum, suggesting an intracranial metastasis.

Due to the aggressive infiltration of the gliomas, migration of tumor cells is not a surprise. For example, if a mass of GBM occurs in one side of the brain, it may try to cross the corpus coliseum into the other half of the brain to make a so-called “Butterfly” sign on MRI scan (bi-hemisphere GBMs) (Figure 2A). which is an almost an unresectable feature for neurosurgeons.

A few decades ago, a chemotherapy agent called Gliadel wafer (Azurity pharmaceutical, Atlanta, GA, USA) went into the market, which contains carmastatin, and is implanted in the brain along the walls and floor of the cavity created after a GBM has been surgically removed. The residual tumor cells felt the threat from the Gliade and started to run away from it. Some tumor cells run through the unhealed surgical wound and form a subcutaneous nodule, with biopsy confirmed, it was GBM. Some surgeons did not like it since it delayed the wound healing, it actually caused by tumor cells running through the surgical wound. In addition, the chemotherapy agent might have some effect on the inhibition of tissue recovery (healing process).

The metastasis of GBM is very rare and only reported as case reports [2]. However, (Figure 2B and C) showed a patient with right temporal GBM, successfully surgical removed; but sometime later, another enhancing nodule showed up on his left cerebellum, suggesting an intracerebral metastatic GBM from the right temporal lobe to left cerebellum.

2. Histopathology of glioblastomas

2.1 Macroscopy

GBMs are often showing signs of elevated intracranial pressure due to the mass effect, while surprisingly large at the time of presentation, and can occupy much of a lobe. Most GBMs of the cerebral hemispheres are clearly intraparenchymal with an epicenter in the white matter (Figure 1D). Those records in the pathology application form by neurosurgeon’s description of a specimen during surgery always include poorly delineated, the cut surface is variable in color, with peripheral grayish tumor masses and central areas of yellowish necrosis. After formalin fixation, GBMs are fragmented and soft, gray to pink rim with peripheral brain tissue. Necrotic or hemorrhagic tissue may also border adjacent brain structures without an intermediate zone of macroscopically detectable tumor tissue. Some of the tumor’s present macroscopic cysts, contain a turbid fluid, and constitute liquefied necrotic tumor tissue (Figure 3).

Figure 3.

Small cell glioblastomas are remarkably uniform in both cell size and distribution. Although the cells are often a bit elongated rather than round, the overall appearance resembles that of lung or other primary small cell carcinomas (A, x200). Epithelioid glioblastomas with plump cytoplasm and sharp cell borders simulate metastatic carcinoma or melanoma. It is difficult to distinguish in some cases, especially intraoperation. BRAF v600E is a marker of characteristic expression in epithelioid glioblastoma, and other immunohistochemistry markers such as HMB45, Melan-a may usually resolve the issue (B, x200). Giant cell glioblastoma is consist predominantly of pleomorphism, multinucleation of large or giant cells, atypical mitoses may be numerous. Sometimes microvascular proliferation is absent (C, x200).

2.2 Cell proliferation

The main cellular feature of malignant glial cells is local tissue invasion that typically occurs along deep white matter tracts. Most GBMs exhibit nuclear atypia, greater cellularity, multiple mitotic figures, and a high degree of nuclear pleomorphism. The neoplastic cells are marked pleomorphism, enlarged hyperchromatic nuclei with clumped chromatin, which is an important histological feature to differentiate astrocytic tumors from oligodendrogliomas. Significant variation in cellularity is often seen in different parts of the tumor and can lead to misdiagnosis if the specimens are obtained by stereotactic needle biopsy [3]. Although most of the cases show readily visible mitoses, the distribution is very unevenly in the same tumor. When pathologists use the Ki-67 proliferation index to evaluate it, different regions could range from 5% to over 70% within a GBM.

2.3 Microvascular proliferation

Since the grading system had been set up at the World Health Organization (WHO) classification of tumors of the central nervous system, microvascular proliferation is the major histological feature of high-grade gliomas, especially at GBMs. The morphology manifests as multilayered small-caliber blood vessels to indicate that they grow rapidly. In some cases, endothelial and smooth muscle cell overgrowth in an organoid structure, so-called “Glomeruloid shape” [4]. In addition to glomeruloid appearance, some remarkably proliferated vessels may be accompanied by necrosis and mitoses [5]. During the intraoperative frozen section, the presence of microvascular proliferation within a hypercellular glial neoplasm is a reliable histological feature to support the diagnosis of a high-grade tumor. As the evidence given by different researches, a number of mechanisms, which include perinecrotic hypoxia, stimulate the growth factor expression, lead to new angiogenesis [6].

2.4 Necrosis

Necrosis is another important histological character in GBM apart from microvascular proliferation. Necrosis in GBM can take on a variety of morphologies, from single tumor cell necrosis to extensive diffuse necrosis, which can be seen under light microscopy. The typical necrosis is the so-called “Pseudopalisading” [7], whereby tumor cells are arranged radially in a picket fence-like distribution around a central area of necrosis. Evidence from other studies suggests that exclusion of microvascular proliferation results in markedly increased of vascular permeability, often with a decrease in microthrombosis. Thrombosis leads to the infarction of surrounding tissues [8]. The relationship between thrombosis and necrosis is much stronger in IDH-wildtype glioblastoma than in IDH-mutant high-grade astrocytomas [9].

2.5 Cytology

The cytologic appearance of GBM is extremely variable and pleomorphic. The background of the smear may be fibrillary, or necrotic, which is helpful to make the diagnosis at intraoperative frozen section. Some cases show an appearance essentially similar to that of low-grade astrocytoma, especially when the surgeon sends a peripheral part of the tumor. Pathologic mitosis, single-cell necrosis, and gradual thinning to dense cell distribution suggest that it is possible to see the boundary region of the tumor. For small cell glioblastoma or very poorly differentiated tumors, cytological features will show up similar to lymphomas or embryonal tumors, at that time spectrum of progressive dedifferentiation, we may find that all intermediate possible aspects, such as cellular anaplastic changes, vascular proliferative changes, and necrotic phenomena add up and combine each other.

2.6 Histological patterns of glioblastoma

GBM is a highly variable morphologic tumor, as the old term “glioblastoma multiforme” mentioned, forming the pivot of the tumor is fusiform, atypia, and pleomorphic cells, but low-grade neoplastic astrocytes are often detectable, more or less. Cellular pleomorphism includes small, undifferentiated, giant, epithelioid, spindled, gemistocytic, lipidized, and sarcomatoid cells. Some tumors may present one kind of pattern dominantly; these can be established in different subtypes of GBMs.

Three main subtypes of GBM are giant cell glioblastoma, epithelioid glioblastoma, and gliosarcoma, each of them has been described in Individual chapters at the World Health Organization (WHO) classification of tumors of the central nervous system since 2016 [10, 11, 12, 13, 14]. In addition to these subtypes, there are several patterns that are characterized by predominant cell type that can be observed in GBM.

Giant cell glioblastoma is histologically characterized by numerous large, bizarre giant cells, which have multiple nuclei and atypical mitosis, small fusiform syncytial cells, and a reticulin background [15]. The giant cells are often extremely bizarre, sometime it can be larger than 0.5 mm in diameter. The pleomorphism shows not only the size of cells, but also multiple nuclei, cytoplasmic inclusions, palisading, and large ischemic necrosis. Giant cell glioblastomas are frequently rich in reticulin, tend to well-circumscribed structure on MRI, and may be diagnosis as a metastasis tumor. The intraoperative consultation of these lesions will be misguided by clinical information, especially when the giant cells spread over carcinoma or melanoma-like pattern. The perivascular accumulation of tumor cells with the formation of a pseudorossettes-like pattern, which is detected in the frozen slides as a typical feature in GBM may be useful for differential diagnosis. Not the same as Non-CNS tumors, most giant cells indicate a poor prognosis, the giant cell subtype of GBM is slightly better in prognosis than that of other ordinary GBM by some studies [16, 17].

Epithelioid glioblastomas are dominated by a relatively uniform population of discohesive rounded epithelioid cells with eccentric nuclei and abundant eosinophilic cytoplasm, distinct cell membrane, paucity of cytoplasmic processes, and laterally positioned nucleus. Tumor cells can display features of squamous or adenomatous epithelial cells, and are immunoreactive to cytokeratin by IHC stain, when it contains keratin pearls or typical glandular structures that will mimic metastatic carcinoma [18, 19]. Rosenthal fibers and eosinophilic granular bodies are not features in this type of tumor and the necrosis is usually showing zonal type compared with ordinary GBM. The pleomorphic xanthoastrocytoma and epithelioid GBM are BRAF p.V600E positive tumors and they will share similar histology, molecular tests will be more important for differential diagnoses [20].

Gliosarcomas are a special subtype of GBM with the biphasic component, which can either present glial or spindled sarcomata’s morphology. The glial part of the mixture is astrocytic, showing features about GBM, and the mesenchymal part of the tumor is most manifesting as spindled fibroblast-like sarcoma. Sometimes the glial component includes epithelial differentiation, such as glandular, adenoid, and squamous formation. The mesenchymal component may be variable, like bone, cartilage, osteoid-chondroid tissue, smooth, and striated muscle, and even lipomatous features could be seen in the tumor [21– 23].

Stains can be useful to distinguish different components of the tumor for the sarcomatous part is rich in collagen and reticulin, which can be seen in the well-developed intensely staining network around spindle cells, and the glial component is seen as reticulin-free nests, which are immunoreactive for GFAP (Figure 4B) [24].

Figure 4.

(H&E 200x) PNET-like pattern in GBM may have a similar cell morphology to those of medulloblastomas and neuroblastoma (A). BRAF is positive in epithelioid tumor cells (B, IHC stain, X200).

GBM is one of the most morphologically heterogeneous tumors, there are several histological patterns that can be detected if a particular cellular morphology predominates besides three main subtypes. Gemistocytic regions in GBMs are similar to other astrocytic neoplasms, which reveal the distinctive cells with large eosinophilic, plump to slightly angulated cytoplasm, and eccentric nuclei. Perivascular lymphocytic infiltrates appear to be more common in this variant. Oligodendrocyte-like cells with uniform round nuclei and variable perinuclear haloes may be seen in some GBMs, including a chichen wire-like capillary network and microcalcifications, suggestive of a presence of low-grade glioma, like secondary GBM. Previous studies suggest that such tumors have a better prognosis than ordinary GBMs, but since evidence from molecular tests prompts that like the outdated name oligoastrocytomas often referred as “mixed glioma” with two components, GBMs with oligodendroglial cells are molecularly heterogeneous. Since 2016, only IDH-wild-type tumor with this pattern is classified as GBM based on the WHO classification [25]. Small cells with highly monomorphic, round to oval, hyperchromatic nuclei, and minimal discernible cytoplasm, which is similar to the small cell neuroendocrine tumor of other organs can be a predominant feature of GBM, as referred as “small cell GBM”, which is with a very poor prognosis [26]. The mitotic activity is vibrant and the Ki-67 index proliferation index is very high in this component. Granular cells which is large, periodic acid Schiff positive cytoplasm can be observed in some cases, occasionally, GBM may be composed of granular cells dominantly. Some granular cells are positive for CD68, but negative CD163, which is easily misinterpreted as macrophage lesion, but that lesion has a distinct histological appearance and is characterized by aggressive clinical behavior [27]. Lipidized cells with foamy cytoplasm are another pattern of GBM. The cells may be grossly enlarged; adipose tissue-like tumor cells may be lobules or diffuse patterns [25]. GBMs with a primitive neuroectodermal tumor (PNET) component present a nest of immature cells with markedly increased cellularity, high N/C ratios, and active mitotic figures. The nodular cells differentiated into neuronal, medulloblastoma-like, even showing Homer-Wright rosettes, and the anaplastic cytology of that is similar to CNS embryonal tumors. These tumors were reported that had increased frequency of cerebrospinal fluid dissemination, like Ewing sarcoma (Figure 5) [28].

Figure 5.

A mixture of gliomatous and sarcomatous tissues in gliosarcoma. There are many inflammatory cells infiltration in the background (A, H&E x200). (B) the GFAP IHC stain highlights glioma components of gliosarcoma (IHC 200x).

3. Molecular genetic bases of GBMs

At molecular level, GBM has at least two subtypes, primary and secondary. In 1996, Watanabe et al. first reported the evidence that primary and secondary GBMs were with distinct genetic alterations [29]. TP53 mutations were found to be uncommon in primary GBMs but occurred more commonly in secondary GBMs. EGFR overexpression was primarily in primary GBMs but was rare in secondary GBMs. Further studies showed TP53, and IDH1 mutation and EGFR overexpression are mutually exclusive events, suggesting two different genetic pathways in the development of GBMs [29]. This hypothesis was further confirmed by additional studies, which provided additional evidence that primary and secondary GBMs develop through distinct molecular pathways [28, 30]. Typical for primary GBMs are EGFR amplification or mutation, PTEN mutation, and entire loss of chromosome 10 [28, 30]; while genetic alterations more common in secondary GBMs include TP53 and IDH1 mutations and 19q loss [28, 30]. Especially, the IDH1 mutation is currently considered as the most characteristic change for the secondary GBMs, as well as those lower-grade gliomas, including both astrocytomas and oligodendrogliomas (Figure 6).

Primary GBM occurs in elderly patients with no history of previous existing lower-grade gliomas, and the tumor is driven by amplification of EGFR and/or mutation of EGFRvIII, while the secondary GBM, the patients had a history of low-grade gliomas and the tumor is under the mutations of IDH1, and p53.

3.1 1p/19q co-deletion

The loss of chromosome arms 1p and 19q is an established genetic hallmark of oligodendroglial tumors; it can be detected in up to 80% of oligodendrogliomas (WHO grade II) and up to 80% in anaplastic oligodendrogliomas (WHO grade III) by a few large scale studies [29, 30].

The co-deletion of 1p/19q has been shown its great prognostic value as the tumors with this type of co-deletion respond much better to chemotherapy, which led to a better prognosis and a longer tumor-free survival time. The co-deletion is not only associated with the patient’s age, but also the tumor’s anatomic locations. For age, the younger the patient, the higher chance of co-deletion. Tumors in frontal lobes carry the highest percentage of co-deletion, followed by the parietal lobe, and occipital lobe, while tumors in the temporal lobe is with the lowest chance of co-deletion. In addition, morphologically the tumor is more typical to the oligodendroglioma, it has more chance to have co-deletion. If the tumor has only one deletion, 1p deletion appears clinically more important than 19q deletion in some early studies. It should be noted that research demonstrated that at least 5% astrocytic neoplasms, including GBMs also have this type of chromosomal deletion, and the astrocytic neoplasms with the co-deletion have shown the same response clinically as the oligodendrogliomas, with better prognosis, better chemotherapy response, and longer tumor-free survival time. Therefore, the test of 1p/19q co-deletion becomes a part of the routine supplementary test in GBM diagnosis, since nowadays, the glial tumor diagnosis requests molecular analysis in our practice, as oncologists request those results for making a treatment plan. Various techniques are available to detect 1p/19q co-deletion; however, fluorescent in situ hybridization (FISH) is often used in many laboratories due to its technical ease, and this type of co-deletion involved the entirely loss of the short arm of chromosome 1 and the long arm of chromosome 19, which makes the FISH test an easy approach (Figure 7). FISH is a pathologist’s favored method in practice, and can be used directly on formalin-fixed and paraffin-embedded tissue and does not require additional tissue from the patient. Another frequently used method is loss of heterozygosity (LOH), which is a PCR-based test that compares tumor DNA to the patient’s “normal” DNA as a control, usually from peripheral blood.

Figure 6.

Picture of glioblastoma is composed of sinuous and hypercellular band of cells, which traces the border of necrotic zones in what is known as pseudopalisading (A, H&E x200). Necrosis in GBM involves both tumor cells and blood vessels. Necrosis in GBMs does not necessarily have pseudopalisading. Either type of necrosis serves WHO 4 tumors as grading criterion (B, H&E x200). Glomeruloid vascular proliferation is a classic histological feature in GBM, multilayered intravascular endothelial cells gathering together (C, H&E x 200).

Figure 7.

1p/19q codeletion by FISH (A, 1p deletion red; B, 19q deletion red).

3.2 IDH mutations

First identifiend in 2008, isocitrate dehydrogenases 1 and 2, (IDH1 and IDH2), are homologous, NADP+ − dependent cytoplasmic and mitochondrial enzymes, respectively. The function of these enzymes is the conversion of Isocitrate to α-ketoglutarate with the simultaneous reduction of NADP+ to NADPH. IDH1 has recently been discovered to be mutated in a vast majority of astrocytic and oligodendroglial neoplasms with WHO grade 2–3, as well as in secondary GBM (WHO grade 4). IDH 1 mutation is very rare in primary GBM and has not been detected in pediatric pilocytic astrocytomas (WHO grade 1).

The most common mutation is heterozygous point mutation with substitution of arginine by histidine at codon 132 (R132H), located in the substrate-binding site. This IDH 1-R132H mutation has a reported rate of 50–93% in gliomas. IDH1 mutation is currently considered the initial step of tumorigenesis in glial neoplasms, including both astrocytic and oligodendral gliomas, although the IDH1 mutation-related gliomagenesis is not fully understood, it appears to be multifactorial. The product and byproduct of the reaction, α-ketoglutarate, and NADPH, both defend against cellular oxidative stress. Therefore, with decreased quantities of these compounds, the cell may be more susceptible to oxidative damage. In addition to the tumorogenetic property conferred by the inability to perform the conversion, it appears that the IDH1 mutation confers an enzymatic gain of function. With the IDH1 mutations, the cancer cell has the gained ability to convert α-ketoglutarate into 2-hydroxyglutarate (2HG). This reaction will not only further decrease α-ketoglutarate store, but will also reduce NADPH to NADP+, further increasing the cell’s susceptibility to oxidative stress. The overproduction of 2HG in the brain has been oncogenic with an increased risk of brain tumors. Furthermore, there is an association between the IDH1 mutation and increases hypoxia-induced factor-1α. Hypoxia-induced factor-1α is a transcription factor associated with tumorigenesis, such as the upregulation of vascular endothelial growth factor, and stimulating tumor angiogenesis. Interestingly, as a matter of factor, vascular proliferation is one of the histopathological features of GBM.

IDH-wild-type GBMs show a widespread anatomical distribution, while IDH-mutant GBMs favor the frontal lobe, which offers the surgeons more wildly resection of the tumors and provides the potential for a better prognosis. In addition, those IDH-mutant gliomas, no matter lower-grade astrocytomas or oligodendrogliomas with 1p/19q co-deletion all favor this location, supporting the hypothesis that these gliomas develop from a distinct population of common precursor cells [14].

IDH1 mutation has been shown to be a strong, independent prognostic biomarker not only in GBMs, but also in diffuse gliomas of lesser grades (grade 2 or 3) as well. There is no difference yet to be seen in terms of the point mutation, R132H verse others, regarding patients’ outcome. While the IDH1 mutation conveys a better patients’ outcome, unlike 1p/19q co-deletion, it does not predict a better response of the glioma to chemotherapy. In addition to its prognostic value, the identification of IDH mutations could be used diagnostically to determine tumor verse reactive conditions. Analysis of IDH1/2 mutations could be utilized in the separation of primary and secondary GBMs and for the challenging cases of differentiating pilocytic astrocytoma from cystic GBM.

Recently, IHC staining by using a specific antibody against mutant IDH1-R132H was developed, which can be applied to routine paraffin-embedded tissue. This has been proved to be a tumor-specific marker differentiating reactive from neoplastic cells in grade II and III gliomas. However, selecting a good antibody is important for practice, since some antibodies on the market lack the sensitivity and specificity requested by pathological diagnosis. In addition, detection of IDH1/2 mutations can also be achieved by PCR techniques and direct sequencing.

Key points:

• Associated with a better outcome and younger age.

• Found in secondary glioblastoma, rarely in primary.

• Also found in grades II-III diffuse gliomas.

• Used for prognosis and diagnosis.

By 2016, WHO classification of tumors of the central nervous system [14], GBM was separated into IDH-wild-type and IDH mutant subtypes based on the mutation status of IDH1/2 genes that encode Isocitrate dehydrogenases 1 and 2 (IDH1 and IDH2).

It was requested as part on the diagnosis of gliomas currently. Adult diffuse gliomas (used to be grade II or III) have at least three molecular subtypes by new WHO classification of tumors of CNS [1]. The first, tumors with IDH mutation and 1p/19q co-deletion, this type tumors are more likely with oligodendroglial differentiation and good prognosis. The second are those tumors with IDH mutation and p53 mutation, are likely with astrocytic differentiation and slightly better prognosis. The third one are those tumors with IDH wild type and more likely astrocytic differentiation and higher grade with poor prognosis. For example, a brain mass biopsied shows infiltrating astrocytoma with active mitoses but no definitive histological features of necrosis and vascular proliferation. IDH1 status was negative by IHC stain and PCR, the tumor was with EGFR amplification and TERT promoter mutation. Despite the histologic absence of tumor necrosis and microvascular proliferation (traditionally diagnosed as grade 2 or 3 astrocytoma), this molecular profile is now considered to be in keeping with an IDH-wild type GBM (CNS WHO grade 4) by the 2021/5th edition of WHO Classification of CNS Tumors [1].

3.3 P53

P53 was one of the first identified tumor suppress genes and is involved in many neoplasms, from carcinomas of lung and breast, sarcomas, to brain tumors. TP53 gene is located on the short arm of chromosome 17. The major function of P53 is to control cell cycle progression, promote apoptosis, DNA integrity, and the survival of cells exposed to DNA damaging agents. In an activated status, P53 acts as a transcription regulator leading to the upregulation of p21. The protein P21 is the stop protein responsible for binding to the cyclin-dependent kinase and inhibiting cell proliferation. Thus, a mutated p53 will be unable to prevent cell replication, resulting in uncontrolled tumor growth.

In most human cancers, PT53 is inactivated by gene alteration, which results in the loss of the protein’s tumor suppressor function.

The majority of mutations involving p53 lead to missense mutations, and there is a resultant prolongation of the protein half-life, which accumulates in the nucleus of the cells. Therefore, by IHC stains for P53 highlight the nuclei of the cells and are used as a surrogate marker for identifying cells affected by a mutation in this pathway.

The significance of the detection of P53 overexpression in gliomas is inconsistent. Some reports indicated that diffused positive of nuclear PT53 stain might correlate IDH1 mutation in secondary GBMs. In terms of diagnosis, p53 would be a less favorable marker than others in distinguishing primary from secondary GBMs given that P53 overexpression has been reported in up to 25% of primary GBMs. As a prognostic marker, p53 has been shown inconsistent results. While some reports indicate a shorter survival time for gliomas overexpressing P53, this finding has not been confirmed by several meta-analyses yet.

3.4 EGFR

EGFR is one of the well-unknown tumor growth factors receptor, and which is involved in many malignancies, from carcinomas of the lung and breast to uncommon sarcomas. The receptor tyrosine kinase (RTK) of EGFR is frequently altered in IDH-wildtype GBM. Overall, about 60% of tumors show evidence of EGFR amplification, mutation, rearrangement, or altered splicing. The most frequent of these alterations is EGFR amplification., which occurs in about 40% of IDH-wild type GBMs and in 60% of GBMs in the DNA methylation group. In the majority of cases, EGFR amplification is associated with a second EGFR alteration, such as extracellular domain mutations or in-frame intragenic deletions encoding either EGFRvIII or other alternative transcripts [1]. Like most growth factor receptors, it is composed of three major parts, an extracellular ligand-binding domain, a transmembrane domain, and an intracellular domain with tyrosine kinase activity. Each those tumor carries a different EGFR mutation. In primary GBM, besides EGFR amplification, the EGFR mutation is characterized by in-frame deletion of exons 2–7, resulting in a truncated extracellular domain with the inability to bind a ligand but retains ligand-independent tonic and constitutive activities to stimulate the tumor nuclei to promote tumor cell proliferation. This mutation is named as EGFRvIII (EGFR variant III), which plays an important role in tumorigenesis by activating Mitogen Active Protient Kinase (MAPK) and phosphoinositide-3-kinase (PI3K-Akt) pathways, leading to cell proliferation, decreased apoptosis, angiogenesis, and aggressive tumor invasion [31]. In most primary GBMs, EGFR amplification and mutation occur simultaneously, which offers the tumor cells a great proliferation advantage, aggressive clinical behavior as well a bad prognosis. EGFR amplification and mutation can be detected by FISH (Figure 8) as well as PCR techniques.

Figure 8.

Amplification of EGFR by FISH (red color) is one of characteristic molecular changes of primary GBM.

3.5 PTEN alteration and 10q LOHs

Phosphatase and tensin homolog (PTEN), located at 10q23, is a tumor suppressor gene with a role in opposing the PI3K-Akt pathway. In gliomas with a mutant PTEN gene, there is an associated increase in PI3-Akt pathway signaling, which may contribute to the tumor’s malignant behavior of aggressively invasion and infiltration. Mutations at the PTEN gene are found in 15–40% of primary GBMs but are absent in IDH1 mutated secondary GBMs and other lower-grade gliomas.

PTEN mutation and 10q LOH both carry the same negative prognostication for GBMs. LOH analysis or FISH can be used for this type of mutation evaluation [31].

Loss of heterozygosity (LOH) at chromosome 10q23 occurs commonly in a different type of human tumors. In GBMs, approximately 70% of GBMs are with PTEN alterations. PTEN is a negative regulator of the phosphoinositide 3 kinase pathway, a major signaling stimulating cellular proliferation in response to growth factor stimulation. PTEN deletions were more common in GBMs, but not in lower-grade, like grade II/III gliomas. PTEN deletion was very common across all gene expression subtypes, but absent in IDH1 mutant tumors [32]. PTEN loss was associated with AKT pathway activity [33]. Several studies demonstrated that patients with loss of PTEN generally had shorter survival than patients with PTEN retention, However, PTEN loss was not associated with worse survival in newly diagnosed GBMs patients of the TMZ era [34].

3.6 TERT promoter mutation in GBMs

Telomerase reverse transcriptase (TERT) in gliomagenesis has been recently further strengthened by the frequent occurrence of TERT promoter mutations (TERTp-mut) in gliomas and many other malignant neoplasms.

The telomerase reverse transcriptase (TERT) gene encodes a highly specialized reverse transcriptase, which adds hexamer repeats to the 3′ end of chromosomes. The increased telomerase activity seen in cancer leads to the preservation of telomeres, allowing tumors to avoid induction of apoptosis.

The promoter region of TERT contains two hotspots for point mutation; with most GBM (about 80% in one study) carry these mutations. They are more common in IDH1-wild type GBMs but rare in secondary (IDH1 mutant) GBMs and other astrocytomas. TERT mutation are also common in oligodendrogliomas. TERTp-mutation is associated with poor outcomes in patients with GBM [35]. A study found that about 75% GBMs were associated with TERTp-mutation, TERTp-mut was associated with IDH-wt, EGFR amplification, CDKN2A deletion, and chromosome 10q loss, but not with MGMT promoter methylation (Combined analysis). TERTp-mutation was an independent factor for poor prognosis. TERTp mutation can be detected by sequencing and RT-PCR [35].

3.7 MGMT status

Epigenetic gene silencing by DNA methylation is another common mechanism of inactivating genes. The MGMT gene encodes a DNA repair protein and is transcriptional silenced by promoter methylation [1]. The interplay between epigenetic regulation (post-translational modification) and GBM tumorigenesis has several modalities. Epigenetic modifiers can be oncogenic or tumor suppressors affected by genetic alteration of gain and loss-of-function, which results in the disruption of epigenetic regulatory processes by affecting histone modification, DNA methylation, and chromatin remodeling. The MGMT (O⁶-methylguanine-DNAethyltransferase) gene at 10q26 encodes for a DNA repair protein. In gliomas of different grades, the MGMT gene is silenced by promoter hypermethylation, impeding transcription, and thus, resulting in a decreased expression of the MGMT protein. This epigenetic modification has been associated with increased sensitivity to alkylating chemotherapy. In alkylating therapies such as temozolomide (TMZ), a methyl group is added to the O⁶-position of the nucleotide guanine, resulting in DNA damage and apoptosis [31]. A full-functioning MGMT would remove this methyl group, however with reduced expression of the protein secondary to promoter hypermethylation the cell has a decreased ability to repair alkylated DNA. Therefore, MGMT expression analysis can be used to predict which tumors may have a more favorable response to alkylating chemotherapeutic agents, like TMZ. Testing of MGMT can be applied to pediatric gliomas as well. MGMT promoter methylation has been found in up to 40% of primary GBMs and 40–60% of secondary GBMs. The aberration is also present in other diffuse gliomas, with a preponderance of oligodendrogliomas at 60–93% [1, 31].

While studies have shown that MGMT promoter methylation results in a significantly longer survival time for patients with GBM treated with concomitant treatment of temozolomide and radiotherapy, there have been discordant reports regarding MGMT methylation as a predictor for increased survival in patients receiving radiotherapy alone. However, in gliomas of lesser grades there is a clear prognostic association between MGMT methylation status and sole radiotherapy. The underlying mechanism by which MGMT methylation would offer a favorable prognosis when not in relation to chemotherapy is a bit more difficult to clarify. As mentioned previously, gliomas often contain multiple molecular aberrancies and thus it may be the result of another molecular change, or the summation of several changes, that convey this prognostic significance to radiotherapy.

The most common method utilized to assess the MGMT promoter methylation status is a methylation-specific PCR analysis, which applies primers composed of differing quantities of CpG sites to allow differentiation between methylated and unmethylated DNA. Methylation-specific pyrosequencing has also been employed with strong sensitivity. Other DNA-based methods are available such as combined bisulfite restriction analysis (COBRA) and methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) [31, 34].

Key points:

• Predictive for a better response of glioblastomas to alkylating chemotherapy.

• Associated with better prognosis in diffuse gliomas treated with radiotherapy, alkylating chemotherapy, or combination therapy.

• Can be found in all glioma types.

In summary, in primary and secondary GBMs, each has its own genetic pathway, which are summarized in the following table for easy reference (Table 1).

Cell of origin
EGFR ampl/mutation	IDH mutation
PETN mutation	p53 mutation
Monosomy10	10q loss
Primary GBM	Secondary GBM

Table 1.

A summary of the major genetic pathway for primary and secondary GBMs.

4. Conclusion

Glioblastoma (GBM) is a malignant tumor of the central nervous system with a very poor prognosis even with current treatment including surgery, chemo, and radiotherapy. Most patients with GBMs have only 15 to 20 months of survival time. In the last two decades, the rapid development of molecular genetic techniques helped us to move our understanding of the GBM into a new level [36, 37]. It is believed that further research will identify new and more important and reliable biomarkers of GBM, which enable us to develop more sensitive target treatment, and eventually, we can overcome this challenging neoplasm.

Abbreviations

GBM	Glioblastoma
FISH	Fluorescent in situ hybridization
IHC	Immunohistochemistry
LOH	Loss of heterozygosity
EGFR	Epidermal growth factor receptor
TMZ	Temozolomide
TERT	Telomerase reverse transcriptase
TERTp-mut	TERT promoter mutation
IDH	Isocitrate dehydrogenase
MGMT	O6-methylguanine-DNA methyltransferase
PTEN	Phosphatase and tensin homolog
NGS	next-generation sequencing
WHO	World Health Organization
CNS	Central nerve system
PNET	primitive neuroectodermal tumor
GFAP	Glial fibrillary acidic protein