**3.7. Glioblastoma IDH-mutant**

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. **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 altera-

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 pos-

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

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

*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 second-

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

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

sible amplifications accompanied by truncation are PDGFRA and MET [127].

at various sites caused by the loss of the chomosomal region [128].

with the methylation of the promoter of the deletion of the CDKN2 gene.

ary glioblastomas than in the primary ones [7].

glioblastomas [131].

promoter, leading to the aberrant expression of the gene [130].

tion in glioblastomas is 7p gain combined with 10q loss [125].

122 Glioma - Contemporary Diagnostic and Therapeutic Approaches

**Definition**. High-grade tumor with a dominantly astrocytic differentiation and a diffuse growth pattern, involving the mutation of the IDH gene.

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

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

**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 enhancing zones on MRI.

**Macroscopy**. The aspect is that of a tumor infiltrating the adjacent cerebral parenchyma. The purple hemorrhagic areas and the yellow-white necrotic ones are absent.

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

*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


**Table 2.** Synthesis of the mutational status in the main categories of diffuse gliomas.

event in tumorigenesis and remains present during the progression toward glioblastoma. The most frequent mutation of the IDH1 gene (90% of all astrocytic or oligodendroglial tumors) is R132H, when a guanine is replaced by an adenine (CGT->CAT). Other mutations, such as R132C, R132S, or R132L, occur much more rarely [137].

The ATRX gene is also mutated, the same mutation being present in the grade II and III precursor astrocytic lesions. Alongside the ATRX and IDH mutation, TP53 is more frequently mutated in secondary glioblastomas [31]. EGFR amplifications are rare, as opposed to the GBM wild-type, indicating that the genetic onset and progression pathways are different.

The genetic expression of GBM IDH-mutant is relatively homogeneous, most of them falling under the proneural profile [126]. Epigenetically, the occurrence of the IDH mutation induces an extensive hypermethylation of the DNA, all IDH-mutant tumors belonging to a hypermethylated phenotype [138]. The prognosis for GBM IDH-mutant is better than that for GBM wild-type, the survival rate being 2.4 times higher (**Table 2**) [139].

### **3.8. Glioblastoma, NOS**

**Definition**. High-grade tumor with dominantly astrocytic differentiation and diffuse growth pattern, in which the mutational status of the IDH gene cannot be determined. They are grade IV tumors.

glioma, the largest at that time, the median of survival through surgery alone was 7 months, whereas in the group receiving additional radiotherapy the median survival rate was at 15 months [140]. The addition of chemotherapy further improved the survival, but despite the large advancements in research within the last decade, the median survival rate barely increased by no more than 2 months, inexorably close to the data reported 20 years ago [141]. Regarding the role of surgery, there is an increasing body of literature underlining the importance of GTR comparing with STR or biopsy. A meta-analysis covering 41,117 unique patients in 37 retrospective studies revealed a significant improvement of OS in GTR cases compared with STR [142]. Another prospective study comparing grade of resection with the aid of immunofluorescence (5-ALA) versus white light also demonstrated a significant median of survival in favor of those with GTR (16.7 months vs. 11.7 months) [143]. Currently, there is sufficient data favoring GTR to encourage surgeons to increase the grade of resection, while also striv-

**Figure 21.** MRI examination of a patient with left paraventricular glioblastoma: (a) T2W sequence reveals an inhomogeneous lesion adjacent to occipital horn of left lateral ventricle surrounded by a hyperintense region of edema; (b) T1W + C sequence expose classical aspect of "ring enhancement"; (c) DWI study demonstrating a restricted diffusion corresponding to the region of tumor; MRI perfusion reveals a heterogeneous rCBV (d) and increased cerebral blood flow (CBF) corresponding to the enhanced lesion (e) ; (f) Tractography shows a disruption of fibers compared to the

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New tools were introduced in the last two decades in order to facilitate GTR, simultaneously with the permanent improvement of the operative microscope. Contemporary neuronavigation

ing not to cause additional neurological deficits (**Figure 21**).

contralateral side.

### *3.8.1. Multimodal treatment*

### *3.8.1.1. Surgical treatment*

Sir Rickman Godlee was the first surgeon who reported a resection of a glioma in 1884. More than one century later, the results of the treatment in malignant gliomas remained unsatisfactory. Improvements of surgical techniques and technology developed in this period, including microsurgery, neuronavigation, intraoperative MRI, intraoperative neuromonitoring, and 5-ALA merely improved the grade of resection while increasing the postoperative quality of life of the patient; however, they were unable to change the inexorable course of malignant gliomas to recurrence and death. Perhaps the most important adjuvant of surgery was the introduction of radiotherapy in the middle of the last century, adding a median survival of at least 7 months, as it was highlighted by Ley and coworkers in 1962. In their reported series of

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event in tumorigenesis and remains present during the progression toward glioblastoma. The most frequent mutation of the IDH1 gene (90% of all astrocytic or oligodendroglial tumors) is R132H, when a guanine is replaced by an adenine (CGT->CAT). Other mutations, such as

**Astrocytoma Oligodendroglioma GBM-wt**

IDH + + - ATRX + - - TERT - + + p53 + - + 1p/19q - + -

The ATRX gene is also mutated, the same mutation being present in the grade II and III precursor astrocytic lesions. Alongside the ATRX and IDH mutation, TP53 is more frequently mutated in secondary glioblastomas [31]. EGFR amplifications are rare, as opposed to the GBM wild-type, indicating that the genetic onset and progression pathways are different.

The genetic expression of GBM IDH-mutant is relatively homogeneous, most of them falling under the proneural profile [126]. Epigenetically, the occurrence of the IDH mutation induces an extensive hypermethylation of the DNA, all IDH-mutant tumors belonging to a hypermethylated phenotype [138]. The prognosis for GBM IDH-mutant is better than that for GBM

**Definition**. High-grade tumor with dominantly astrocytic differentiation and diffuse growth pattern, in which the mutational status of the IDH gene cannot be determined. They are grade

Sir Rickman Godlee was the first surgeon who reported a resection of a glioma in 1884. More than one century later, the results of the treatment in malignant gliomas remained unsatisfactory. Improvements of surgical techniques and technology developed in this period, including microsurgery, neuronavigation, intraoperative MRI, intraoperative neuromonitoring, and 5-ALA merely improved the grade of resection while increasing the postoperative quality of life of the patient; however, they were unable to change the inexorable course of malignant gliomas to recurrence and death. Perhaps the most important adjuvant of surgery was the introduction of radiotherapy in the middle of the last century, adding a median survival of at least 7 months, as it was highlighted by Ley and coworkers in 1962. In their reported series of

R132C, R132S, or R132L, occur much more rarely [137].

124 Glioma - Contemporary Diagnostic and Therapeutic Approaches

**Table 2.** Synthesis of the mutational status in the main categories of diffuse gliomas.

wild-type, the survival rate being 2.4 times higher (**Table 2**) [139].

**3.8. Glioblastoma, NOS**

*3.8.1. Multimodal treatment*

*3.8.1.1. Surgical treatment*

IV tumors.

**Figure 21.** MRI examination of a patient with left paraventricular glioblastoma: (a) T2W sequence reveals an inhomogeneous lesion adjacent to occipital horn of left lateral ventricle surrounded by a hyperintense region of edema; (b) T1W + C sequence expose classical aspect of "ring enhancement"; (c) DWI study demonstrating a restricted diffusion corresponding to the region of tumor; MRI perfusion reveals a heterogeneous rCBV (d) and increased cerebral blood flow (CBF) corresponding to the enhanced lesion (e) ; (f) Tractography shows a disruption of fibers compared to the contralateral side.

glioma, the largest at that time, the median of survival through surgery alone was 7 months, whereas in the group receiving additional radiotherapy the median survival rate was at 15 months [140]. The addition of chemotherapy further improved the survival, but despite the large advancements in research within the last decade, the median survival rate barely increased by no more than 2 months, inexorably close to the data reported 20 years ago [141].

Regarding the role of surgery, there is an increasing body of literature underlining the importance of GTR comparing with STR or biopsy. A meta-analysis covering 41,117 unique patients in 37 retrospective studies revealed a significant improvement of OS in GTR cases compared with STR [142]. Another prospective study comparing grade of resection with the aid of immunofluorescence (5-ALA) versus white light also demonstrated a significant median of survival in favor of those with GTR (16.7 months vs. 11.7 months) [143]. Currently, there is sufficient data favoring GTR to encourage surgeons to increase the grade of resection, while also striving not to cause additional neurological deficits (**Figure 21**).

New tools were introduced in the last two decades in order to facilitate GTR, simultaneously with the permanent improvement of the operative microscope. Contemporary neuronavigation

benefit of 2–3 months in the group treated with combined radio-chemotherapy and this combination has been the standard of care for almost 25 years [147]. In time, whole brain radiotherapy was replaced by a more focal radiotherapy, in order to prevent secondary effects of radionecrosis. The current standard of radiotherapy is a fractionated conformational dose of 2Gy fractions/day, 5 days/week for a period of 6 weeks. The radiotherapy regimen must be

**Figure 23.** Ultrasonography-guided resection (a) of a diffuse astrocytoma with foci of glioblastoma (arrow) demonstrated

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Concerning chemotherapy, the actual standard consists of concomitant radiotherapy and temozolomide (TMZ) administration, followed by six cycles of TMZ at every 28 days. This was established in 2005 based on the results of the trial conducted by Stupp et al. (**Figure 23**) [79]. The response of the patients to the TMZ regimen is highly variable, with one of the factors influencing the positive response being the methylation of the MGMT promoter, which is present in less than 50% of glioblastoma cases. It was showed that only 65–70% of new cases of glioblastoma respond to TMZ; although in spite of these evidences, TMZ is still the main

A large number of researches were made in order to decrypt the intimate mechanism of apparition and development of glioblastomas, as well as the response of tumor cells to different chemical, physical, or biological agents. Among these agents, only few met the clinical criteria to be introduced in practice. Gliadel (carmustine-impregnated biodegradable wafers) on local application proved to add a median of survival of 2.3 months compared to the standard treatment, yet was accompanied by an increased incidence of local complications (infection and CSF fistula) [150]. We also add our research effort on glioblastoma stem cell cultures in order to improve the response to TMZ by addition of new drugs like arsenic trioxide or metformin as sensitizers [151, 152]. New ways to deliver TMZ have been proposed [153]. Antiangiogenic agents (Bevacizumab) was also tested on glioblastoma stem cells with controversial response

The clinical trials testing Bevacizumab in newly diagnosed glioblastoma failed to demonstrate a benefit on survival [154, 155]. Immunotherapy is a highly experimental approach of present days, as are other therapies. A special interest was raised in the use of an electric field delivered by noninvasive transducers placed on a headpiece, the so-called Novocure

initiated in three to no more than 6 weeks after the surgery [148].

on T1W + C image (b).

agent in multimodal treatment of glioblastoma (**Figure 24**) [149].

(in some cultures, they increased angiogenesis) (**Figure 25**).

**Figure 22.** Preoperative (a and b) and postoperative (c and d) contrasted CT scan images demonstrating the GTR of a left gliosarcoma.

equipment offers a more precise localization with real-time correction adapted to the brain shift. Functional and anatomical data are merged in order to facilitate surgical intervention, while also avoiding damage to eloquent areas and tracts. Intraoperative tools, such as intraoperative MRI, are now available in many centers, allowing surgeons to achieve a more complete tumor removal and, as a direct consequence, prolong survival [144]. Intraoperative ultrasonography, having been introduced in practice two decades prior, is at present more accurate in defining intracerebral lesions, especially in cases of HGG, facilitating a real-time control of resection (**Figure 22**) [145].

Contrasted intraoperative ultrasound guidance apparently adds more detailed information concerning the grade of resection [146].

We may conclude that surgery, and radical surgery especially, remains the first and most important step of the multimodal treatment in prolonging the survival of patients with malignant gliomas.

### *3.8.1.2. Adjuvant treatment*

Whole brain radiation therapy stood as the most important adjuvant to surgery up until the randomized trial conducted by Walker el al. in 1980. In this trial, the authors compared the efficacy of radiotherapy alone to the addition of nitrosourea chemotherapy. They demonstrated a

**Figure 23.** Ultrasonography-guided resection (a) of a diffuse astrocytoma with foci of glioblastoma (arrow) demonstrated on T1W + C image (b).

benefit of 2–3 months in the group treated with combined radio-chemotherapy and this combination has been the standard of care for almost 25 years [147]. In time, whole brain radiotherapy was replaced by a more focal radiotherapy, in order to prevent secondary effects of radionecrosis. The current standard of radiotherapy is a fractionated conformational dose of 2Gy fractions/day, 5 days/week for a period of 6 weeks. The radiotherapy regimen must be initiated in three to no more than 6 weeks after the surgery [148].

Concerning chemotherapy, the actual standard consists of concomitant radiotherapy and temozolomide (TMZ) administration, followed by six cycles of TMZ at every 28 days. This was established in 2005 based on the results of the trial conducted by Stupp et al. (**Figure 23**) [79].

The response of the patients to the TMZ regimen is highly variable, with one of the factors influencing the positive response being the methylation of the MGMT promoter, which is present in less than 50% of glioblastoma cases. It was showed that only 65–70% of new cases of glioblastoma respond to TMZ; although in spite of these evidences, TMZ is still the main agent in multimodal treatment of glioblastoma (**Figure 24**) [149].

equipment offers a more precise localization with real-time correction adapted to the brain shift. Functional and anatomical data are merged in order to facilitate surgical intervention, while also avoiding damage to eloquent areas and tracts. Intraoperative tools, such as intraoperative MRI, are now available in many centers, allowing surgeons to achieve a more complete tumor removal and, as a direct consequence, prolong survival [144]. Intraoperative ultrasonography, having been introduced in practice two decades prior, is at present more accurate in defining intracerebral lesions, especially in cases of HGG, facilitating a real-time

**Figure 22.** Preoperative (a and b) and postoperative (c and d) contrasted CT scan images demonstrating the GTR of a

Contrasted intraoperative ultrasound guidance apparently adds more detailed information

We may conclude that surgery, and radical surgery especially, remains the first and most important step of the multimodal treatment in prolonging the survival of patients with malignant gliomas.

Whole brain radiation therapy stood as the most important adjuvant to surgery up until the randomized trial conducted by Walker el al. in 1980. In this trial, the authors compared the efficacy of radiotherapy alone to the addition of nitrosourea chemotherapy. They demonstrated a

control of resection (**Figure 22**) [145].

126 Glioma - Contemporary Diagnostic and Therapeutic Approaches

concerning the grade of resection [146].

*3.8.1.2. Adjuvant treatment*

left gliosarcoma.

A large number of researches were made in order to decrypt the intimate mechanism of apparition and development of glioblastomas, as well as the response of tumor cells to different chemical, physical, or biological agents. Among these agents, only few met the clinical criteria to be introduced in practice. Gliadel (carmustine-impregnated biodegradable wafers) on local application proved to add a median of survival of 2.3 months compared to the standard treatment, yet was accompanied by an increased incidence of local complications (infection and CSF fistula) [150]. We also add our research effort on glioblastoma stem cell cultures in order to improve the response to TMZ by addition of new drugs like arsenic trioxide or metformin as sensitizers [151, 152]. New ways to deliver TMZ have been proposed [153]. Antiangiogenic agents (Bevacizumab) was also tested on glioblastoma stem cells with controversial response (in some cultures, they increased angiogenesis) (**Figure 25**).

The clinical trials testing Bevacizumab in newly diagnosed glioblastoma failed to demonstrate a benefit on survival [154, 155]. Immunotherapy is a highly experimental approach of present days, as are other therapies. A special interest was raised in the use of an electric field delivered by noninvasive transducers placed on a headpiece, the so-called Novocure

**3.9. Diffuse midline glioma, H3 K27 M-mutant**

onset of other more common clinical signs [157].

could be detected in few cases (**Figure 26**).

areas or yellowish-white necrotic ones.

**Definition**. High-grade infiltrative astrocytic tumor, with midline location and showing the

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*Clinically*, it is usually encountered in children and more rarely in adults. The symptoms may be either general (usually produced by the obstructive hydrocephalus) or focal generated by the involvement of different cranial nerves: facial paresis, diplopia, hearing loss, dysphonia, and dysphagia. The extension of the tumor in cerebellar peduncles produces ataxia. The involvement of long tracts determines motor weakness. Particularly in diffuse pontine glioma, behavioral changes such as pathological laughter and anxiety were described before the

**Imaging**. On MRI, these lesions have a diffuse homogenous appearance of hypointense in T1W sequences and hyperintense in T2W and FLAIR ones. The contrast is discrete or absent, but some focal enhancement into an infiltrative hypointense mass on T1W + C sequences

*MRI spectroscopy* will differentiate these tumors from other inflammatory, vascular, or infectious lesions. The Cho/Cr and NAA/Cr ratios are typically increased. An elevation of these

**Macroscopy**. The infiltrative pattern is reflected in the size and the changes in the shape of the nervous structures affected by the tumor. In cross section, we can see purple hemorrhagic

**Histological diagnosis**. The aspect is that of an intensely infiltrative tumor consisting of small-size monomorphic, astrocytic cells. While most are accompanied by necrosis, microvascular proliferations, and an enhanced mitotic index, these elements are absent in a small number of tumors. According to the new rule introduced by the latest WHO classification, whereby "molecular beats histology," even if histologically they are grade II, they shall nevertheless be deemed grade IV. *Immunohistochemistry*. GFAP shows a heterogeneous expression in these tumors. On the other hand, they are positive for S100, OLIG2, and NCAM1. Mutations of ATRX and TP53 can occur, but more rarely. An antibody targeting the protein modified by the presence of the H3.3 K27 M mutation can be used in the diagnosis [159].

**Figure 26.** In vitro 3D model of glioblastoma stem cells culture demonstrating an increase angiogenesis in contact with

TMZ and resistance to the addition of Bevacizumab compared with the control (ctrl) (personal archive).

ratios after radiotherapy poses the significance of progression [158].

K27 M mutation in the H3F3A or HIST1H3B/C genes. They are grade IV tumors.

**Figure 24.** Preoperative axial (a), coronal (b) and sagittal (c) T1W + C sequences of a patient with glioblastoma completely removed and treated with the standard radio-chemotherapy regimen; postoperative enhanced T1W images (d–f) reveal the removal of the tumor with the persistence of a small contrasted nodule, which is stable at 5 years after surgery.

**Figure 25.** Preoperative T1W + C sequence (a) of a glioblastoma, almost completely removed as it was revealed by immediate postoperative CT scan (b); the patient was not responsive to the standard adjuvant treatment as it is demonstrated by the postoperative enhanced MRI at 4 months (c).

treatment. The addition of this new modality to the standard radio-chemotherapy regimen improved the PFS and OS in glioblastoma-treated patients [156].

We can conclude that despite the huge efforts and investments made in research and therapies, the results still disappoint. This suggests the fact that we are possibly on the correct course, but against the tide. The future multimodality treatment may consist of patient-tailored treatments owing to the multiple individual and tumor-related factors that influence the response to therapy.
