**3.6. Glioblastoma IDH wild-type**

**Definition**. High-grade tumor with a diffuse growth pattern and a dominantly astrocytic differentiation showing cellular pleomorphism, nuclear atypia, and brisk mitotic activity. Glioblastomas are characterized by the presence of necrosis and microvascular proliferations.

**Grading**. Glioblastomas are grade IV WHO.

*Clinically*, the evolution is usually short due to the high growth rate of the tumor [90]. Almost 40% of the patients are admitted for surgical treatment in the first month from the clinical debut marked by progressive headache (almost 80% of cases). Mental disturbances, changes in personality, and neurological deficits are subsequent manifestations of the evolution of the disease. Seizures are less common (18.9% in our study on 276 cases) then in the LGG [81].

**Imaging**. *Native CT scan* is usually performed in emergency and reveals a heterogeneous expansive process with significant mass effect. Addition of contrast enhancement clearly defines a variable-enhanced lesion with perilesional edema, the classical aspect of a ring enhancement circumscribing an necrotic area which is frequently present (**Figure 18**).

Advanced MRI techniques are also useful for differentiating between local recurrence and radionecrosis (pseudoprogression). Recently, it was showed that rCBV as a single examination modality is superior to volume transfer constant (Ktrans) or apparent diffusion coefficient (ADC) for the prediction of local recurrence. The combination of rCBV and Ktrans

**Figure 18.** Axial T2W (a), axial FLAIR (b) and coronal T1W + C (c) sequences of an extremely rare intraventricular anaplastic oligodendroglioma completely removed, as it is demonstrated by the 6 months follow-up MRI (d–f).

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**Macroscopy**. Poorly delineated lesions, with yellow, white, or granular areas caused by the necrosis, surrounded by grayish tumoral areas. In glioblastomas, cysts filled with a turbid liquid are formed through the liquefaction of the necrotic tissue. Typically, in cross section, we see purple-red or brown-red areas caused by the hemorrhage occurred during the progression of the tumor. The hemorrhages can be massive. Also in cross section, we can see

**Microscopic diagnosis**. The histology of the tumor is extremely variable and the cellular composition is highly heterogeneous. The astrocytic nature of the tumor can be identified in the well-differentiated tumors. In the poorly-differentiated ones, the astrocytic phenotype is rec-

There are a number of cellular morphologies common to glioblastomas: fibrillary astrocytes, gemistocytes, smalls cells (highly infiltrative, bipolar, and mitotically active), epithelioid,

improves the accuracy of the recurrence diagnosis [94].

thrombotic vessels with the aspect of "dark veins."

ognized with considerable difficulty [95].

Once the suspicion brain tumor is raised, an *MRI examination* will more accurately define the lesion. T1W and T2W sequences are able to reveal the tumor and also other pathological changes within or around it: edema, necrosis, hemorrhage, and calcifications. In addition, the FLAIR sequences will demonstrate a hyperintense perilesional halo, which is in fact a combination of tumoral infiltration and edema. On enhanced T1W sequences, the classical appearance of "ring enhancement" delineating a central necrosis is the most eloquent aspect of glioblastoma (**Figure 19**).

Nevertheless, *Advanced MRI* techniques will offer functional elements in favor of a malignant lesion. On MR spectroscopy, glioblastoma presents higher Cho (Choline) peaks along with a higher Cho/Cr (creatine) ratio. This ratio is additionally elevated owing to the decrease of Cr compared with the normal level. Lactate and lipid peaks that indicate necrosis and disruption of myelin sheaths could also be detected in glioblastomas [91]. On DWI, high-grade gliomas typically present a restricted diffusion. DTI studies may help in differentiating HGG from LGG trough fractional anisotropy (FA), which is higher in the former [92]. On tractography, HGG will show a disruption of tracts. MRI perfusion studies reveal a heterogeneous relative cerebral volume (rCBV), demonstrating areas of increased cellularity and mitotic activity in glioblastomas (**Figure 20**) [93].

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Due to the good response of anaplastic oligodendrogliomas to chemotherapy, the subtotal resection with preservation of neurological function is also an acceptable surgical strategy [88].

Adjuvant treatment is recommended in anaplastic oligodendrogliomas on a regular basis. Recent studies failed to demonstrate a benefit of temozolomide over the classical PCV regimen, which remains the main tool for first-line chemotherapy in anaplastic oligodendrogliomas. Radiotherapy is reserved for cases of clear imagistic evidence of tumor progress. Fractions of 1.8–2 Gy for a total dose of 54–60 Gy are the actual recommended radiotherapy

**Definition**. High-grade tumor with a diffuse growth pattern and a dominantly astrocytic differentiation showing cellular pleomorphism, nuclear atypia, and brisk mitotic activity. Glioblastomas are characterized by the presence of necrosis and microvascular proliferations.

*Clinically*, the evolution is usually short due to the high growth rate of the tumor [90]. Almost 40% of the patients are admitted for surgical treatment in the first month from the clinical debut marked by progressive headache (almost 80% of cases). Mental disturbances, changes in personality, and neurological deficits are subsequent manifestations of the evolution of the disease. Seizures are less common (18.9% in our study on 276 cases) then in the LGG [81]. **Imaging**. *Native CT scan* is usually performed in emergency and reveals a heterogeneous expansive process with significant mass effect. Addition of contrast enhancement clearly defines a variable-enhanced lesion with perilesional edema, the classical aspect of a ring

enhancement circumscribing an necrotic area which is frequently present (**Figure 18**).

delineating a central necrosis is the most eloquent aspect of glioblastoma (**Figure 19**).

Once the suspicion brain tumor is raised, an *MRI examination* will more accurately define the lesion. T1W and T2W sequences are able to reveal the tumor and also other pathological changes within or around it: edema, necrosis, hemorrhage, and calcifications. In addition, the FLAIR sequences will demonstrate a hyperintense perilesional halo, which is in fact a combination of tumoral infiltration and edema. On enhanced T1W sequences, the classical appearance of "ring enhancement"

Nevertheless, *Advanced MRI* techniques will offer functional elements in favor of a malignant lesion. On MR spectroscopy, glioblastoma presents higher Cho (Choline) peaks along with a higher Cho/Cr (creatine) ratio. This ratio is additionally elevated owing to the decrease of Cr compared with the normal level. Lactate and lipid peaks that indicate necrosis and disruption of myelin sheaths could also be detected in glioblastomas [91]. On DWI, high-grade gliomas typically present a restricted diffusion. DTI studies may help in differentiating HGG from LGG trough fractional anisotropy (FA), which is higher in the former [92]. On tractography, HGG will show a disruption of tracts. MRI perfusion studies reveal a heterogeneous relative cerebral volume (rCBV), demonstrating areas of increased cellularity and mitotic activity in

*3.5.1.1. Adjuvant treatment*

regimen based on clinical evidence [89].

116 Glioma - Contemporary Diagnostic and Therapeutic Approaches

**Grading**. Glioblastomas are grade IV WHO.

**3.6. Glioblastoma IDH wild-type**

glioblastomas (**Figure 20**) [93].

**Figure 18.** Axial T2W (a), axial FLAIR (b) and coronal T1W + C (c) sequences of an extremely rare intraventricular anaplastic oligodendroglioma completely removed, as it is demonstrated by the 6 months follow-up MRI (d–f).

Advanced MRI techniques are also useful for differentiating between local recurrence and radionecrosis (pseudoprogression). Recently, it was showed that rCBV as a single examination modality is superior to volume transfer constant (Ktrans) or apparent diffusion coefficient (ADC) for the prediction of local recurrence. The combination of rCBV and Ktrans improves the accuracy of the recurrence diagnosis [94].

**Macroscopy**. Poorly delineated lesions, with yellow, white, or granular areas caused by the necrosis, surrounded by grayish tumoral areas. In glioblastomas, cysts filled with a turbid liquid are formed through the liquefaction of the necrotic tissue. Typically, in cross section, we see purple-red or brown-red areas caused by the hemorrhage occurred during the progression of the tumor. The hemorrhages can be massive. Also in cross section, we can see thrombotic vessels with the aspect of "dark veins."

**Microscopic diagnosis**. The histology of the tumor is extremely variable and the cellular composition is highly heterogeneous. The astrocytic nature of the tumor can be identified in the well-differentiated tumors. In the poorly-differentiated ones, the astrocytic phenotype is recognized with considerable difficulty [95].

There are a number of cellular morphologies common to glioblastomas: fibrillary astrocytes, gemistocytes, smalls cells (highly infiltrative, bipolar, and mitotically active), epithelioid,

rhabdoid, granular, or lipidized cells. The areas that contain astrocytes whose phenotype is more easily identified can be more or less clearly separated from the areas of high pleomorphism. The cells are poorly differentiated, with pleomorphism, significant atypia, and brisk mitotic activity. The presence of a cellular population having a different phenotype indicates

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The dominance of a specific cell type in the tumor generates a pattern that is useful in the diagnosis of small cells, with a neuroectodermal component, with an oligodendroglial component, with granular cells, with gemistocytes, and with lipidized cells. This is an indicative

*The small cell glioblastoma pattern* consists of small, round, or slightly oblong monomorphic cells, faint atypia, and hyperchomatic nuclei surrounded by a small amount of cytoplasm. One characteristic is the heightened proliferative activity. The GFAP is variably expressed, and when present, it can be seen at the level of delicate processes. Several morphological elements found in this pattern, such as the monomorphic nuclei surrounded by a clear halo, chicken wire vasculature, or calcifications, can cause problems in the sense of a differential diagnosis with oligodendrogliomas [97]. The difference is made here by molecular tests, which evidence a molecular profile typical for glioblastomas: EGFR amplifications, Ch 10

*The primitive neuronal component glioblastoma* is a glioblastoma featuring nodules that show neuronal differentiation. These nodules are clearly distinguishable from the rest of the tumor. Cellularity is increased, and so is the proliferation index in these areas. The cells are similar to those in other CNS embryonal neoplasms or they form Homer-Wright rosettes. The immunohistochemical profile is closer to that of neuronal precursors, being positive for Synaptophysin

*The glioblastoma with an oligodendroglioma component* contains foci (often minor) of classic oligodendroglioma, associated with necrosis. The presence of the two components in the same tumor, in association with necrosis, raises the possibility of a grade IV oligoastrocytoma. While the presence and the size of the necrosis means a less favorable prognosis, this type of tumor, nevertheless, has a better prognosis than the standard glioblastomas [100]. From a molecular point of view, this histopathological aspect includes several molecular groups, lacking a distinct molecular signature. The tumor may or may not feature IDH1 mutations or 1p/19q codeletion, and also TP53 mutations, Chr 7 gain, Chr 10 loss, EGFR, and PDGFRA amplifications [101].

*Granular cells glioblastomas* are tumors with a distinct histological aspect, characterized by an aggressive behavior, despite a morphological character indicative of a grade II or III tumor. The cells are large, similar to macrophages. They are positive for CD163 and CD68 due to the high lysosome content. They are generally negative for GFAP, even if a certain positivity can be occasionally seen in the peripheral cellular areas. They are little understood from a molecular point of view, but a study involving array-based comparative genomic hybridization has indicated a genetic profile very similar to the conventional glioblastomas (gain of CHr 7, loss of Chr. 1p, 8p, 9p, 10p, 13q, and 22q, EGFR amplification, and homozygous deletion of

CDKN2A). There are no IDH mutations and no 1p/19q codeletion [102].

the emergence of a new tumoral clone through new genetic events [96].

for the diagnosis but insufficient for a distinct variant.

losses, IDH wild-type, no 1p/19q codeletion [98].

and with a low GFAP expression [99].

**Figure 19.** Coronal (a) and axial (b) preoperative contrasted CT scan examination of a case with left temporal inhomogeneous expansive lesion with a "ring enhancement" highly suggestive for a glioblastoma; (c) and (d) immediate postoperative contrasted CT scan examination demonstrating gross total removal of the tumor.

**Figure 20.** T2W (a), FLAIR (b) and T1W + C MRI (c) sequences of a left frontal expansive lesion, aspects that are highly suggestive for a glioblastoma.

rhabdoid, granular, or lipidized cells. The areas that contain astrocytes whose phenotype is more easily identified can be more or less clearly separated from the areas of high pleomorphism. The cells are poorly differentiated, with pleomorphism, significant atypia, and brisk mitotic activity. The presence of a cellular population having a different phenotype indicates the emergence of a new tumoral clone through new genetic events [96].

The dominance of a specific cell type in the tumor generates a pattern that is useful in the diagnosis of small cells, with a neuroectodermal component, with an oligodendroglial component, with granular cells, with gemistocytes, and with lipidized cells. This is an indicative for the diagnosis but insufficient for a distinct variant.

*The small cell glioblastoma pattern* consists of small, round, or slightly oblong monomorphic cells, faint atypia, and hyperchomatic nuclei surrounded by a small amount of cytoplasm. One characteristic is the heightened proliferative activity. The GFAP is variably expressed, and when present, it can be seen at the level of delicate processes. Several morphological elements found in this pattern, such as the monomorphic nuclei surrounded by a clear halo, chicken wire vasculature, or calcifications, can cause problems in the sense of a differential diagnosis with oligodendrogliomas [97]. The difference is made here by molecular tests, which evidence a molecular profile typical for glioblastomas: EGFR amplifications, Ch 10 losses, IDH wild-type, no 1p/19q codeletion [98].

*The primitive neuronal component glioblastoma* is a glioblastoma featuring nodules that show neuronal differentiation. These nodules are clearly distinguishable from the rest of the tumor. Cellularity is increased, and so is the proliferation index in these areas. The cells are similar to those in other CNS embryonal neoplasms or they form Homer-Wright rosettes. The immunohistochemical profile is closer to that of neuronal precursors, being positive for Synaptophysin and with a low GFAP expression [99].

**Figure 19.** Coronal (a) and axial (b) preoperative contrasted CT scan examination of a case with left temporal inhomogeneous expansive lesion with a "ring enhancement" highly suggestive for a glioblastoma; (c) and (d) immediate

**Figure 20.** T2W (a), FLAIR (b) and T1W + C MRI (c) sequences of a left frontal expansive lesion, aspects that are highly

postoperative contrasted CT scan examination demonstrating gross total removal of the tumor.

118 Glioma - Contemporary Diagnostic and Therapeutic Approaches

suggestive for a glioblastoma.

*The glioblastoma with an oligodendroglioma component* contains foci (often minor) of classic oligodendroglioma, associated with necrosis. The presence of the two components in the same tumor, in association with necrosis, raises the possibility of a grade IV oligoastrocytoma. While the presence and the size of the necrosis means a less favorable prognosis, this type of tumor, nevertheless, has a better prognosis than the standard glioblastomas [100]. From a molecular point of view, this histopathological aspect includes several molecular groups, lacking a distinct molecular signature. The tumor may or may not feature IDH1 mutations or 1p/19q codeletion, and also TP53 mutations, Chr 7 gain, Chr 10 loss, EGFR, and PDGFRA amplifications [101].

*Granular cells glioblastomas* are tumors with a distinct histological aspect, characterized by an aggressive behavior, despite a morphological character indicative of a grade II or III tumor. The cells are large, similar to macrophages. They are positive for CD163 and CD68 due to the high lysosome content. They are generally negative for GFAP, even if a certain positivity can be occasionally seen in the peripheral cellular areas. They are little understood from a molecular point of view, but a study involving array-based comparative genomic hybridization has indicated a genetic profile very similar to the conventional glioblastomas (gain of CHr 7, loss of Chr. 1p, 8p, 9p, 10p, 13q, and 22q, EGFR amplification, and homozygous deletion of CDKN2A). There are no IDH mutations and no 1p/19q codeletion [102].

*The lipidized glioblastoma* occurs rarely, a significant number of large cells with a lipidized (foamy) aspect being present against a histological background typical for a standard glioblastoma. In such cases, the diagnosis of pleomorphic xanthoastrocytoma cannot be ruled out. From a genetic point of view, they are little studied [103]. Phenotypically, they are similar to adipocytes, but the fact that they are positive for GFAP comes to confirm their glial origin.

structures in the mesenchymal component (but not in the astrocytic one) can be demonstrated using staining for reticulin and trichrome. As with the giant cell glioblastomas, the mesenchymal component makes the tumor macroscopically similar to a metastasis or meningioma. This entity can also appear in ependymomas and oligodendrogliomas, de novo or following treatment [111]. Immunohistochemically, the astrocytic component is positive for GFAP, while the sarcomatoid one is negative. P53 is positive in both components. IDHR132H is negative. Genetically, this variant shows PTEN and TP53 mutations, as well as CDK2A deletions. There are no EGFR amplifications [113]. The epithelial-mesenchymal transition in the sarcomatoid

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The microvascular proliferations are in direct relation with the necrosis, hypoxia being a significant factor that stimulates the formation of vessels by way of HIF1A and VEGFA [114]. Another characteristic is the thrombosis of small vessels. Although, in glioblastomas, vascularization is very well represented, the vascular structures are immature and largely incapable

Necrosis, of the coagulative type, is typical for glioblastoma. It affects both the cells and the vascular structures and is indicative of the extremely high proliferation rate associated with this type of tumor. It is considered to be an aggressive factor in astrocytic tumors, its size being associated with a lower survival rate. In the necrotic areas, we can see "shadows" of the dilated vessels surrounded by tumor cells in various stages of decay [116]. We can also see vessels that still contain oxygenated blood surrounded by viable cells. Undoubtedly, the high proliferation rate of glioblastomas plays an important part in the onset of necrosis, as there is a mismatch between the need for oxygen and the number of functioning vessels. As in the vicinity of the necrotic areas, we notice venal occlusions and vascular thrombosis, and it has been hypothesized that this is the mechanism that could trigger or spread the necrosis. Lesions of the endothelium trigger the release of procoagulants, which generate microscopic or macroscopic vascular thrombosis. Vascular thrombosis is usually accompanied by the socalled pseudopalisading [25]. This is caused by the migration of cells from the central necrotic area to the more oxygenated exterior ones, in a "moving away wave" toward the vessels unaffected by thrombosis and capable of maintaining a level of oxygenation sufficient for their survival. In an attempt to somewhat restore the level of oxygenation, these cells also secrete proangiogenic factors (VEGFs), causing the vascular alterations mentioned above [117].

A perivascular positioning of lymphocytes can occur in the areas with gemistocytes. In glioblastomas, the number of lymphocytes can vary, and they are mainly LT CD8. Quite interestingly, the presence of an extensive LT CD8 infiltration has been identified with the long-term survivors. LT CD4 and LB are also present, but in small numbers. Microglia and histiocytes can also be seen [118]. Immunohistochemically, GFAP has a positive expression, its variability reflecting the heterogeneous nature of the tumor. Sarcomatous and primitive cellular components are negative, while the gemistocytic component is strongly positive. It is also positive in the lipidized variant. S100 and Olig2 are positive in glioblastomas and are quite useful in the diagnosis of poorly differentiated tumors [119]. Nestin is of particular importance in the differential diagnosis in

component is highlighted by the SNAIL and TWIST expression.

Necrosis and microvascular proliferations are required for a diagnosis.

of compensating for the hypoxia caused by the exuberant proliferation [115].

Three histological variants are known: giant cell glioblastoma, gliosarcoma, and epithelioid glioblastoma.

*Giant cell glioblastomas* are characterized by the occurrence of giant multinucleated cells (up to 20 nuclei), with prominent nucleoli. Also noticeable are the small syncytial fusiform cells. The infiltration patterns typical for the diffuse gliomas are less visible in this variant. It features a significant component of connective tissue in the shape of a reticulin network [104]. Atypical mitoses are frequent. Another characteristic feature of this variant is the perivascular positioning of tumor cells (pseudorosette-like pattern). There is a significant ischemic necrosis. Microvascular proliferation and the pseudopalisading necrosis are rarely encountered. Perivascular lymphocytes may be present [105]. The neuronal immunohistochemical markers are negative. GFAP has variable expression. The nuclear expression of p53 shows a high incidence of TP53 gene mutation [106]. The presence of the connective component, together with their circumscribed nature, makes it more difficult to distinguish macroscopically between these tumors and meningiomas and metastases.

From a molecular point of view, they showed a high incidence of PTEN and TP53 mutations; but, no EGFR amplifications and no homozygous deletion of CDK2A were shown. As a variant of the wild-type glioblastoma, it features no IDH mutations. The prognosis is slightly better than in the case of usual glioblastomas [107].

*The epithelioid glioblastoma* is characterized by the presence of a significant population of cells with an epithelioid aspect, eosinophilic cytoplasm, peripheral nucleus, and sharp border. Rhabdoid cells with discrete borders and eccentric oval nuclei can be present, as well as necrosis and vascular proliferations. Immunohistochemically, the GFAP has a variable expression [108]. The epithelial markers (EMA and CK AE1/AE5), as well as S100 and vimentin, are positive. The antibody that indicates the BRAF V600 mutation is positive in 50% of cases [109]. From a molecular point of view, the epithelioid glioblastoma occasionally shows genetic events characteristic of glioblastomas (EGFR amplifications, CKD2A deletion, and PTEN mutation). As a variant of the wild-type glioblastoma, it features no IDH mutations. The prognosis is worse than in the case of other glioblastomas [110].

*Gliosarcoma* is characterized by a monoclonal proliferation with a highly malignant biphasic histological pattern, astrocytic and mesenchymal. The astrocytic component displays the typical aspect of a glioblastoma. The mesenchymal component consists of long bundles of spindle cells, closely packed, showing malignancy (atypia, necrosis, and mitosis), similar to fibrosarcoma or the malignant fibrous histiocytoma [111]. The confirmation of malignancy in the mesenchymal component is required in order to differentiate it from desmoplasia, which can occur in the glioblastomas with meningeal invasion. Elements of differentiation can appear (cartilage, bone, muscle, and osteoclastic giant cells) [112]. The presence of connective tissue structures in the mesenchymal component (but not in the astrocytic one) can be demonstrated using staining for reticulin and trichrome. As with the giant cell glioblastomas, the mesenchymal component makes the tumor macroscopically similar to a metastasis or meningioma. This entity can also appear in ependymomas and oligodendrogliomas, de novo or following treatment [111]. Immunohistochemically, the astrocytic component is positive for GFAP, while the sarcomatoid one is negative. P53 is positive in both components. IDHR132H is negative. Genetically, this variant shows PTEN and TP53 mutations, as well as CDK2A deletions. There are no EGFR amplifications [113]. The epithelial-mesenchymal transition in the sarcomatoid component is highlighted by the SNAIL and TWIST expression.

Necrosis and microvascular proliferations are required for a diagnosis.

*The lipidized glioblastoma* occurs rarely, a significant number of large cells with a lipidized (foamy) aspect being present against a histological background typical for a standard glioblastoma. In such cases, the diagnosis of pleomorphic xanthoastrocytoma cannot be ruled out. From a genetic point of view, they are little studied [103]. Phenotypically, they are similar to adipocytes, but the fact that they are positive for GFAP comes to confirm their glial origin. Three histological variants are known: giant cell glioblastoma, gliosarcoma, and epithelioid

*Giant cell glioblastomas* are characterized by the occurrence of giant multinucleated cells (up to 20 nuclei), with prominent nucleoli. Also noticeable are the small syncytial fusiform cells. The infiltration patterns typical for the diffuse gliomas are less visible in this variant. It features a significant component of connective tissue in the shape of a reticulin network [104]. Atypical mitoses are frequent. Another characteristic feature of this variant is the perivascular positioning of tumor cells (pseudorosette-like pattern). There is a significant ischemic necrosis. Microvascular proliferation and the pseudopalisading necrosis are rarely encountered. Perivascular lymphocytes may be present [105]. The neuronal immunohistochemical markers are negative. GFAP has variable expression. The nuclear expression of p53 shows a high incidence of TP53 gene mutation [106]. The presence of the connective component, together with their circumscribed nature, makes it more difficult to distinguish macroscopically between

From a molecular point of view, they showed a high incidence of PTEN and TP53 mutations; but, no EGFR amplifications and no homozygous deletion of CDK2A were shown. As a variant of the wild-type glioblastoma, it features no IDH mutations. The prognosis is slightly

*The epithelioid glioblastoma* is characterized by the presence of a significant population of cells with an epithelioid aspect, eosinophilic cytoplasm, peripheral nucleus, and sharp border. Rhabdoid cells with discrete borders and eccentric oval nuclei can be present, as well as necrosis and vascular proliferations. Immunohistochemically, the GFAP has a variable expression [108]. The epithelial markers (EMA and CK AE1/AE5), as well as S100 and vimentin, are positive. The antibody that indicates the BRAF V600 mutation is positive in 50% of cases [109]. From a molecular point of view, the epithelioid glioblastoma occasionally shows genetic events characteristic of glioblastomas (EGFR amplifications, CKD2A deletion, and PTEN mutation). As a variant of the wild-type glioblastoma, it features no IDH mutations.

*Gliosarcoma* is characterized by a monoclonal proliferation with a highly malignant biphasic histological pattern, astrocytic and mesenchymal. The astrocytic component displays the typical aspect of a glioblastoma. The mesenchymal component consists of long bundles of spindle cells, closely packed, showing malignancy (atypia, necrosis, and mitosis), similar to fibrosarcoma or the malignant fibrous histiocytoma [111]. The confirmation of malignancy in the mesenchymal component is required in order to differentiate it from desmoplasia, which can occur in the glioblastomas with meningeal invasion. Elements of differentiation can appear (cartilage, bone, muscle, and osteoclastic giant cells) [112]. The presence of connective tissue

glioblastoma.

these tumors and meningiomas and metastases.

120 Glioma - Contemporary Diagnostic and Therapeutic Approaches

better than in the case of usual glioblastomas [107].

The prognosis is worse than in the case of other glioblastomas [110].

The microvascular proliferations are in direct relation with the necrosis, hypoxia being a significant factor that stimulates the formation of vessels by way of HIF1A and VEGFA [114]. Another characteristic is the thrombosis of small vessels. Although, in glioblastomas, vascularization is very well represented, the vascular structures are immature and largely incapable of compensating for the hypoxia caused by the exuberant proliferation [115].

Necrosis, of the coagulative type, is typical for glioblastoma. It affects both the cells and the vascular structures and is indicative of the extremely high proliferation rate associated with this type of tumor. It is considered to be an aggressive factor in astrocytic tumors, its size being associated with a lower survival rate. In the necrotic areas, we can see "shadows" of the dilated vessels surrounded by tumor cells in various stages of decay [116]. We can also see vessels that still contain oxygenated blood surrounded by viable cells. Undoubtedly, the high proliferation rate of glioblastomas plays an important part in the onset of necrosis, as there is a mismatch between the need for oxygen and the number of functioning vessels. As in the vicinity of the necrotic areas, we notice venal occlusions and vascular thrombosis, and it has been hypothesized that this is the mechanism that could trigger or spread the necrosis. Lesions of the endothelium trigger the release of procoagulants, which generate microscopic or macroscopic vascular thrombosis. Vascular thrombosis is usually accompanied by the socalled pseudopalisading [25]. This is caused by the migration of cells from the central necrotic area to the more oxygenated exterior ones, in a "moving away wave" toward the vessels unaffected by thrombosis and capable of maintaining a level of oxygenation sufficient for their survival. In an attempt to somewhat restore the level of oxygenation, these cells also secrete proangiogenic factors (VEGFs), causing the vascular alterations mentioned above [117].

A perivascular positioning of lymphocytes can occur in the areas with gemistocytes. In glioblastomas, the number of lymphocytes can vary, and they are mainly LT CD8. Quite interestingly, the presence of an extensive LT CD8 infiltration has been identified with the long-term survivors. LT CD4 and LB are also present, but in small numbers. Microglia and histiocytes can also be seen [118].

Immunohistochemically, GFAP has a positive expression, its variability reflecting the heterogeneous nature of the tumor. Sarcomatous and primitive cellular components are negative, while the gemistocytic component is strongly positive. It is also positive in the lipidized variant. S100 and Olig2 are positive in glioblastomas and are quite useful in the diagnosis of poorly differentiated tumors [119]. Nestin is of particular importance in the differential diagnosis in regard to other high-grade gliomas, as it is positive in glioblastomas [120]. P53 is positive in the glioblastomas with a missense mutation of TP53 [121]. Together with WT1 (which can be positive in low-grade gliomas), it makes the distinction between tumor cells and the reactive posttreatment cells [122]. EGFR indicates the relative amplification of the gene, being expressed in 45–95% of cases. EGFRvIII is present in one third of all cases [123]. The expression of Ki-67 varies. A positive IDH R132H is incompatible with the diagnosis of IDH wild-type glioblastomas.

**Epigenetics**. A study conducted in cooperation with TCGA and based on the determination of the genomic profile has defined four subtypes of glioblastomas: pro-neural, neural, classic, and mesenchymal. These subtypes are characterized by a different mutational and epigenetic profile, reflected in the response to treatment. These results have paved the way toward the assessment of the epigenetic profile of glioblastomas through a determination of the whole methylation genome profile in the glioblastomas with artificially induced mutations (IDH H3F3A). These experiments have led to the identification of six different subtypes, with a different clinical evolution [132].

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MicroRNA and long non-coding RNA have also been studied in glioblastomas. miR10b controls the cycle of stem and tumoral cells in GBM and is associated with a poor prognosis. The role of the interaction between microRNA and the oncogenetic pathways known as drivers in

The currently defined histological entities have different genetic expression profiles, which

The methylation profile of the MGMT gene promoter is predictive for the response to the treatment with alkylants such as temozolomide or methylants. It is variable, being higher in

**Definition**. High-grade tumor with a dominantly astrocytic differentiation and a diffuse

*Clinically* speaking, as it is mostly located in the frontal lobe, it is often accompanied by behavioral and neurocognitive changes. As the growth is more sluggish than in the case of wild-

**Imaging**. The areas of central necrosis typically seen in wild-type GBM are usually absent here. They are larger in size; the cystic structures are more frequent; and they show no enhanc-

**Macroscopy**. The aspect is that of a tumor infiltrating the adjacent cerebral parenchyma. The

**Histological diagnosis**. The morphological aspect of IDH-mutant glioblastomas is very similar to that of wild-type GBM. The areas of necrosis (ischemic or palisading) are more rarely encountered. On the other hand, the oligodendroglioma-like component is more frequent and

*Immunohistochemically*, GFAP is positive and shows a certain variability. The presence of genetic events is reflected by the positivity for IDH1 and p53, and the negativity for ATRX. The overexpression of EGFR is unusual, with amplification being a characteristic of the wild-type GBM [136]. **Genetic diagnosis**. The presence of the IDH mutation falls under the definition of the tumor, and its identification makes it possible to diagnose the two types of glioblastoma. It is an early

type tumors, the signs indicating an increase in intracranial pressure are also milder.

purple hemorrhagic areas and the yellow-white necrotic ones are absent.

has been associated with the presence of the IDH1 mutation [135].

are correlated with the grade and are a better predictor of patient outcome [37].

GBM, such as for instance PI3K, has also been demonstrated [133].

the IDH-mutant tumors, having a G-CIMP profile [134].

growth pattern, involving the mutation of the IDH gene.

**3.7. Glioblastoma IDH-mutant**

ing zones on MRI.

**Grading**. It is ranked as a grade IV tumor.

**Genetic diagnosis**. Glioblastomas were the first tumors investigated by The Cancer Genome Atlas (TCGA), which highlighted alterations in the signaling pathways of EGFR, PDGFR, PI3K, NF1, TP53, and *Rb* [124]. The genetic profile of wild-type glioblastomas differs from that of IHD mutated glioblastomas, which are considered secondary and which have a genetic profile very similar to that of grade II and III astrocytomas. The characteristic genetic alteration in glioblastomas is 7p gain combined with 10q loss [125].

In what concerns the *tyrosine kinase receptors* and their signaling pathways (PI3K/PTEN/AKT/ mTOR and EGFR/RAS/NF1/PTEN/P13K), EGFR is the amplified gene most frequently present in primary glioblastomas, but it is more rarely encountered in the secondary ones [126]. The amplification is accompanied by different truncations in the same tumor. The best known one is EGFRvIII, present in nearly half of the glioblastomas with amplified EGFR. The structure of the receptor is similar to v-erb, and it activates independently of the ligand. Other possible amplifications accompanied by truncation are PDGFRA and MET [127].

The PTEN gene suffers changes almost exclusively in the primary glioblastomas, either by the way of a missense mutation in the area homologous to tensin/auxilin, or following truncation at various sites caused by the loss of the chomosomal region [128].

The *TP53/MDM2/MDM4/p14ARF signaling pathway* is affected in both primary and secondary glioblastomas, especially in the secondary ones, and it is also present in grade II and III astrocytomas. MDM2 amplification is a mechanism whereby the proapoptotic and antiproliferative control of p53 is eluded and encountered in the glioblastomas that do not present TP53 mutations [129].

The CDKN2A locus generates several CDKN2 and p14ARF proteins that act as tumor suppressors. The loss of p14ARF expression is encountered in glioblastomas, being correlated with the methylation of the promoter of the deletion of the CDKN2 gene.

*The CDKN2A/CD4/RB1 signaling pathway* is altered in most glioblastomas and it occurs in both primary and secondary glioblastomas. The mutations of the RB1 gene are rare, and the methylation of the promoter followed by the loss of protein expression is more frequent in secondary glioblastomas than in the primary ones [7].

TERT can show mutations at the level of the promoter, especially in wild-type glioblastomas, being mutually exclusive with TP53. The occurrence of the mutation (in one of the two hot spots) is followed by the accumulation of the GABP transcription factor at the level of the promoter, leading to the aberrant expression of the gene [130].

The IDH gene is not mutated by definition in wild-type glioblastomas, and the evaluation of the mutational status of this gene can make the distinction between primary and secondary glioblastomas [131].

**Epigenetics**. A study conducted in cooperation with TCGA and based on the determination of the genomic profile has defined four subtypes of glioblastomas: pro-neural, neural, classic, and mesenchymal. These subtypes are characterized by a different mutational and epigenetic profile, reflected in the response to treatment. These results have paved the way toward the assessment of the epigenetic profile of glioblastomas through a determination of the whole methylation genome profile in the glioblastomas with artificially induced mutations (IDH H3F3A). These experiments have led to the identification of six different subtypes, with a different clinical evolution [132].

MicroRNA and long non-coding RNA have also been studied in glioblastomas. miR10b controls the cycle of stem and tumoral cells in GBM and is associated with a poor prognosis. The role of the interaction between microRNA and the oncogenetic pathways known as drivers in GBM, such as for instance PI3K, has also been demonstrated [133].

The currently defined histological entities have different genetic expression profiles, which are correlated with the grade and are a better predictor of patient outcome [37].

The methylation profile of the MGMT gene promoter is predictive for the response to the treatment with alkylants such as temozolomide or methylants. It is variable, being higher in the IDH-mutant tumors, having a G-CIMP profile [134].
