Section 1 Glioblastoma

## **Chapter 1** Glioblastoma in Elderly Population

*Raphael Bastianon Santiago, Hamid Borghei-Razavi, Mauricio Mandel, Bhavika Gupta, Asad Ali, Badih Adada and Surabhi Ranjan*

#### **Abstract**

Glioblastoma (GBM) is the third most common primary intracranial tumor and the commonest primary malignant brain tumor in adults. The peak incidence is between 65 and 84 years old. The incidence of GBM increases starkly with age—from 1.3/100,000 between the ages of 35–44 to 15.3/100,000 between the ages of 75–84 years. Elderly patients with GBM have increased comorbidities, lower functional status, aggressive tumor biology, and an overall worse outcome as compared with their younger counterparts. Age is an independent and powerful prognosticator of GBM outcomes, even if the performance status is controlled. Elderly patients with GBM represent a vulnerable heterogeneous cohort. Surgical resection in elderly patients offers a better outcome and improved quality of life as compared with biopsy alone and nowadays can be safely tolerated by elderly patients in specialized centers. The standard of care treatment of glioblastoma based on the Stupp's protocol excluded patients over the age of 70. Thus, the standard of care treatment in elderly patients with GBM remains controversial. Selected elderly patients with excellent performance status may be treated with Stupp's protocol. Elderly patients with lower functional status may be treated with a hypofractionated treatment regimen with concomitant and adjuvant temozolomide. Frail patients with MGMT methylated tumor can be treated with temozolomide monotherapy alone. It is also not unreasonable to treat elderly frail patients with MGMT unmethylated GBM with hypofractionated RT alone. Thus, treatment of elderly patients with GBM needs a multidisciplinary approach based on the extent of the tumor, MGMT methylation status, performance status, and even the social situation unique to the elderly patient. This chapter seeks to bring a comprehensive and updated review on the treatment of glioblastoma in the elderly population.

**Keywords:** glioblastoma, high-grade glioma, elderly, geriatric, hypofractionated, aged, frail, temozolomide, chemoradiation, tumor-treating field

#### **1. Introduction**

Glioblastoma (WHO grade 4) is the third most common primary intracranial tumor and the most common primary malignant brain tumors in adults [1]. While death rates for many common cancers are declining due to prevention (e.g. tobacco control policies) [2], cancer screening [3], and immunotherapy (i.e. lung cancer,

melanoma) [4, 5] and other advances in chemotherapeutics, the prognosis for patients with glioblastoma remains dismal with an overall survival of 12–18 months. The standard of care treatment for glioblastoma is maximum safe resection, followed by combined radiotherapy and temozolomide chemotherapy, and then monthly temozolomide for 6 months [6]. In 2011, a medical device called tumor-treating field was approved to deliver low frequency electromagnetic field locally to the tumor site and was found to further improve the median overall survival by 4.9 months [7].

Though glioblastoma can affect people at any age, it preferentially occurs in older individuals, with a peak incidence between 65 and 84 years old. The 2021 WHO classification of the central nervous system tumors has mandated that the term glioblastoma be used to indicate only IDH wildtype (WT) astrocytoma, WHO grade 4, and *not* IDH-mutant astrocytoma, WHO grade 4 [8]. IDH-WT glioblastoma occurs de novo and its prognosis is much worse than IDH-mutant astrocytoma, WHO grade 4. IDH-mutant astrocytoma, WHO grade 4 were previously also called secondary glioblastoma, and were found in younger patients. The reality is that IDH-wildtype glioblastoma is mostly a disease of the elderly. Yet, there continues to be a lack of clarity and unresolved challenges in treatment of elderly glioblastoma patients leading to a stark contrast in survival outcomes of the elderly (median overall survival of 4 months) vs. non-elderly glioblastoma population (median overall survival 15 months) [9].

What are some unique challenges faced by elderly glioblastoma patients? Elderly patients whose initial symptoms are confusion, memory loss, fatigue or depression are often diagnosed late and have a longer lead time to radiological and pathological diagnosis as compared to patients who present with seizures [10, 11]. Stroke and transient ischemic attacks are common in the elderly population and many glioblastomas are initially misdiagnosed as sub-acute infarcts. This delay in precious lead times often results in a larger tumor size and a worse neurological state at the time of surgery and initiation of treatment. It is well known that patients who undergo resection or de-bulking over a biopsy have better survival outcomes. Yet, elderly GBM patients are more likely to get biopsy over resection due to their frailty, neurological symptom burden, co-morbidities, large tumor size and lower surgical risk tolerance by surgical team and patient families. Therefore, by the time the elderly patient is radiographically and pathologically diagnosed, their condition may have declined too far to be able to tolerate the standard 6 weeks of combined chemoradiotherapy. Their treatment is usually tailored to either hypofractionated chemoradiotherapy, hypofractionated radiotherapy (HFRT) alone or temozolomide alone [12] Even if they are able to get the standard of care 6 weeks concurrent chemoradiation and adjuvant temozolomide, the treatment toxicity is much higher as compared to the non-elderly GBMs [13] and often necessitate treatment discontinuation. The next issue is the uniqueness of tumor biology in elderly glioblastoma. Is it possible that the tumor itself is more aggressive than their non-elderly counterpart? Is the aged brain parenchyma more conducive to tumor growth? Does aging decrease systemic immune-surveillance in the elderly? Further, complex socio-economic factors come in play in regard to treatment access to the elderly. Patients often live by themselves, in assisted living facilities or nursing homes. The treatment of glioblastoma is fully outpatient, thus making it vital that a full-time caregiver be available so that the patient can access the healthcare system. This is often not the case for elderly patients, and it is a not uncommon for them to be diagnosed with GBM and be transitioned to hospice care at the time of initial hospitalization.

In the following book chapter, we closely examine each of the above issues and present the most up-to-date evidence on the unique aspects of glioblastoma in the elderly.

#### **2. Epidemiology**

The incidence of primary brain tumors increases with age [14]. Glioblastoma is the most common malignant brain tumor in the aging population and accounts for 58% of all gliomas in the elderly [15]. The definition of elderly itself is a contested topic. Some researchers consider age 65+ or even 60+ as elderly, while in general most will agree that the population over 70 is elderly. The incidence rate of glioblastoma progressively increases as we grow older—from 1.3/100,000 between the ages of 35–44, 3.6/100,000 between the ages of 45–54, 8.1/100,000 between the ages of 55–64 to the dramatically higher rate of 13/100,000 between the ages of 65–74 and 15.3/100,000 between 75 and 84 years of age [1]. According to a recent study, non-Hispanic whites make up the majority of that population, and males were 1.62 times more likely to be affected than females. The study also concluded that the incidence of glioblastoma remained stable in the past couple of years [16]. Incidence rates for glioblastomas were highest in supratentorial regions and lowest in extra-cranial regions like the spinal cord [1].

#### **3. Signs and symptoms**

In a study on 339 elderly GBM patient over the age 70, the most common presenting symptoms were confusion (38%), hemiparesis (35%), speech disturbance (34%) and seizure (29%) [17]. Another study on 189 elderly GBM patients found that patients most commonly presented with global symptoms of cognitive dysfunction, headache, dizziness and fatigue (66%), followed by loss of neurological function (58%), headache (33%) and seizure (32%) [11]. This study also found if behavioral change, memory impairment and confusion were the presenting symptoms, elderly patients had the shortest overall survival because these symptoms were misinterpreted as normal aging by patients' families and even their healthcare teams. Patients who present with seizures had a significantly longer survival and tend to be younger [11, 18].

#### **4. Prognostic factors**

Age alone is a prognostic factor in GBM [19–21], as elderly patients usually have increased incidence of comorbidities, lower functional status [22, 23] and a unique tumor biology as compared to younger population [21]. In elderly patients with GBM, age and performance status form a complex interplay. The most common performance status assessment tool for primary brain tumors is the Karnofsky Performance Scale (KPS) [24]. A patient with a KPS of 70% is self-caring but is unable to carry on normal activity or do active work. Interestingly, a large study of over 48,000 patients with GBM over the age of 60, found that that even when performance status is good (KPS ≥ 70), overall survival is poorer with advancing age—15.2 months (age 60–69) vs. 9.6 months (age 70–79) vs. 6.8 months (age ≥ 80) [25]. A poor KPS < 70 has also

been associated with a poorer overall survival as patients with a lower KPS usually cannot tolerate a more aggressive treatment (i.e., radical resection followed by chemotherapy and radiotherapy) [26].

#### **5. Tumor biology**

There are molecular, epigenetic, and genomic biomarkers unique to elderly GBMs which are associated with a worse prognosis [19–21, 26]. Elderly GBMs usually lack the isocitrate dehydrogenase (IDH) mutation, which is usually found in younger glioblastoma patients (currently, reclassified as IDH-mutant astrocytoma, WHO grade 4) and is associated with an improved prognosis [12, 27]. It is well established that methylation of DNA repair O(6)methylguanine-DNA methyltransferase (MGMT) is related to response to alkylating agents, and the lack of promoter methylation in this elderly group leads to a poor response to chemotherapy [12, 27, 28]. Fukai et al. in a cohort of 212 patients found that *TERT* promoter mutation, copy number alterations such as *PTEN* deletion and *CDK4* amplification/gain, and co-amplification of *MDM2* and *CDK4* were more frequent in the older group (>70 years old) [20]. There was also a higher triple overlapping of *PTEN*, *CDKN2A* and *EGFR* in the older group, which is positively associated with tumor invasiveness and resistance to therapy [29–31].

As the central nervous system ages, markers of cellular senescence come to the fore [32–34]. One may assume that the senescent cells are growth-arrested and are the polar opposite of oncogenic cells which divide uncontrollably. However, these senescent cells release pro-inflammatory factors called secretory associated senescent phenotype (SASP) which exacerbate cancer. SASP factors such are TGF-beta, IL-6, IL-8, VEGF, matrix metalloprotein-2 (MMP-2) and MMP-9 play a role in diseases such as cancer, neuroinflammation and neurodegeneration [14, 35].

#### **6. Preoperative assessment**

Age alone, even in octogenarian and nonagenarian, should not be the criterion to exclude surgical resection [36–41]. A comprehensive assessment using risk prediction tools for outcomes after a GBM resection in elderly patients is recommended. Patient's performance status is commonly evaluated using KPS (100 = normal to 0 = dead) [24] or the ECOG scale (0 = fully active to 5 = dead) [36] A KPS value lower than 70 is associated with a poor prognosis [21, 38, 39] and is commonly used as a cut-off for patient enrollment for newly diagnosed glioblastoma trials. A more comprehensive appraisal using *Comprehensive Geriatric Assessment* (CGA) which takes in account an older patient's functional status, comorbid medical conditions, cognition, psychological state, social support, nutritional status, and a review of the patient's medications has demonstrated prognostic and predictive role for treatment eligibility [21, 42–44]. CGA is interdisciplinary and multidimensional evaluation that includes eight major criteria (**Figure 1**) to formulate a better plan to anticipate and address challenges in management of geriatric patients. Lombardi et al. in a retrospective study of 133 patients found a prognostic significance for CGA in elderly patients with GBM [43]. Cloney et al. applying *The Canadian Study on Health and Aging Modified Frailty Index (CSHA-mFI)* found that frailer patients, independently of age, KPS or cardiovascular risk, were less likely to undergo surgery, had a longer inpatient stay, had more post-operative complications

#### **Figure 1.**

*Comprehensive geriatric assessment consisting in eight general topics: medical, functional, social, environmental, advanced care, spirituality and sexuality and intimacy. CGA has been advocated for elderly patients with cancer by The International Society of Geriatric Oncology.*

and a shorter overall survival [45]. Thus, in this heterogeneous group a complete and thorough assessment should be performed. While CGA is mainstream in the practice of geriatric oncology, its use is not prevalent in assessment of elderly GBM patients and needs to be encouraged.

#### **7. Treatment overview**

A radical total resection followed by combined temozolomide and standard fractionated radiotherapy (SFRT), followed by adjuvant temozolomide for six cycles, is the standard first line of treatment of glioblastoma. This treatment is based on the EORTC/NCIC study on in which 573 patients after biopsy or resection were treated with 6 weeks of focal radiotherapy (total 60 Gy in 30 fractions) vs. radiotherapy plus continuous daily temozolomide (75 mg/m2 of body-surface area per day, 7 days per week from the first to the last day of radiotherapy), followed by six cycles of adjuvant temozolomide (150–200 mg/m2 for 5 days during each 28-day cycle) [6]. This seminal study found that the median survival in the radiotherapy only group was 12.1 months and was significantly improved to 14.6 months in the combined radiotherapy and chemotherapy group. It is noteworthy that this study excluded patients above the age of 70. Interestingly this study found that in patients >60 years, median survival

was 11.8 months with radiotherapy alone vs. 10.9 months with combination therapy. However, this was an exploratory analysis, and no firm conclusion could be drawn from this subset.

The best treatment approach for elderly glioblastoma patients remains controversial [46]. Due to patient's lower KPS, higher prevalence of risk factors, and the question of ability to tolerate combined standard of care 60Gy chemoradiotherapy, studies on elderly glioblastoma have focused on making the treatment regimens tolerable to patients. Patients with a poor performance status (i.e., KPS < 70) can benefit from temozolomide monotherapy (especially if *MGMT* promoter methylated) or radiotherapy alone [27, 28, 46–50]. The specific dose of radiotherapy adopted is usually different from standard protocol, as the standard radiotherapy dose of 60 Gy can be difficult to tolerate, especially when combined with temozolomide [28]. In this context, hypofractioned radiotherapy (HFRT) has shown not inferior to SFTR with less side effects [51]. Another important factor to consider is that drugs for symptomatic management such as corticosteroids and antiepileptic drugs as they are less tolerated in this group [47]. Thus, there is a need to tailor the therapy for each individual patient's profile in this age group.

#### **8. Surgical treatment**

Some authors have questioned if age alone should be the criterion to decide whether elderly GBM patients, especially those who are 80+, should undergo a surgical resection. The surgical question on elderly GBM patients is two-pronged. First, can the elderly tolerate a major brain surgery or biopsy similar to their nonelderly counterparts? And second, does resection as opposed to biopsy only, confer a survival benefit in elderly patients similar to non-elderly patients? The hesitation for craniotomies on elderly GBM patients, hinges on the fact that octogenarians have an increased incidence of comorbidities and lower functional status, and therefore may not be good candidates for a major surgery [52]. The higher prevalence of metabolic, neurologic, cardiac comorbidities and a loss of reserve capacity seen in this age-group is associated with a lengthier hospitalization [53].

The first question on the safety and tolerance of brain surgery in elderly is answered by several retrospective studies. In a retrospective cohort study of 741 patients with surgically assessable brain tumors, of whom 570 patients were between the ages of 60 and 79 (senior) and 83 were aged 80 or above (elderly), pre- and postoperative change to modified Rankin score, surgical complications, length of stay, and 30-days readmission were performed [36]. No statistical significance was found comparing the elderly patients with their counterparts of senior and young (20– 29 years) (surgical complication rates of 6, 7.2 and 4.5% respectively). Post-operative complications such as neurological deficits, infection, DVTs are similar to those described in younger patients [10, 54, 55]. Thus, it appears that surgical resection in elderly can be safely performed in specialized centers without overt risk as compared to the non-elderly population.

The second question on the benefit of surgical resection as opposed to biopsy stems from the fact that elderly GBMs inherently have a more aggressive biology [19, 21, 35] and that the survival benefit from a radical resection seen in younger GBM patients may not translate to elderly GBM patients. This answer was explored by Chaichana et al. in a retrospective study comparing biopsy in 40 elderly GBM (65 years and older) patients to resection in 40 matched elderly GBM patients [56]. *Glioblastoma in Elderly Population DOI: http://dx.doi.org/10.5772/intechopen.106408*

Overall survival in the resection group (5.7 months) was significantly greater than the biopsy group (4 months). Surgical resection offers a better outcome and is associated with an improved quality of life [57, 58] than biopsy in elderly with GBM [26, 59–62]. Gross total resection (GTR) is related to longer survival time, progression free survival and improved functional recovery without increased morbidity or mortality, when compared to subtotal resection [10, 63, 64].

#### **9. Post-operative assessment**

Length of stay (LOS) has been shown to be longer in the elderly who undergo surgery. There is a positive correlation with LOS and delirium in aged patient [65, 66]. In a study highlighting the incidence of delirium in the elderly, it was found that patients with advanced age had a higher rate of post-operative delirium (POD) and postoperative cognitive dysfunction (POCD) [67, 68]. A possible approach to dealing with POD in the elderly is to optimize pharmacologic intervention. Antipsychotic regimen and use of dexmedetomidine may reduce post-operative delirium and are possible options for pharmacologic interventions to reduce LOS [69–71]. Other factors that could influence the LOS difference between elder and younger groups are mechanical factors such as early ambulation and the use of physical therapy. Chiu et al. found that additional factors such as geriatric consultation, care giver education, and music therapy can also play an important role in decreasing LOS [72].

#### **10. Radiotherapy**

The importance of SFRT in the treatment of glioblastoma has been established more than a decade ago [6]. The time to initiate radiation treatment is between 3 and 6 weeks after surgery [6, 73]. The standard course of 60Gy divided in 30 fractions is widely used in management of glioblastoma, although it is associated with a higher incidence of radionecrosis [74], and may not be well-tolerated in elderly population [12]. Hypofractionated radiotherapy has been shown to be non-inferior to standard radiotherapy in elderly patients. A randomized phase III trial by the Nordic Clinical Brain Tumor Study Group, enrolled patients over 60 years of age in three arms temozolomide only, standard course of radiotherapy (60 Gy, 30 fractions) or hypofractionated radiotherapy HFRT) (34 Gy, 10 fractions) [12]. There was no cut-off for performance status so that real-world scenario could be replicated. For patients aged 70 and older, outcomes were worse in the standard radiotherapy group. For temozolomide vs. hypofractionated radiotherapy, median survival was similar. Only 72% of patients in the standard radiotherapy group could complete their treatment as opposed to 94% patients in the hypofractionated group. If elderly patients had difficulty tolerating 6 weeks of radiation only, then it may be extrapolated that tolerance would be much worse if they were to receive the combined 6 weeks radiation plus concomitant temozolomide. However, this may not apply to fitter elderly patients with KPS 70 or more. A randomized trial of 695 patients testing tumor-treating fields (TTF) after standard chemoradiotherapy and adjuvant temozolomide included 134 patients aged 65 or older and KPS of 70 or higher [7]. In this group of relatively fit elderly patients, the median overall survival was 13.7 months in the adjuvant temozolomide group vs. 17.4 months in the adjuvant temozolomide plus TTF group. Though this study did not discuss the tolerability of standard chemoradiation in the

elderly population, the median survival of over 12 months in the each of the elderly group suggests that elderly patients with a good KPS can in fact tolerate standard 6 weeks of chemoradiation.

HFRT schedules were developed in an effort to improve treatment tolerability and decrease the daily burden of radiotherapy treatment for elderly. In a pre-temozolomide era prospective study on 100 GBM patients, 60 years and older were treated with either HFRT (40 Gy in 15 fractions) vs. standard radiotherapy (60 Gy in 30 fractions) [75]. Overall survival was similar in HFRT group (5.6 months) as compared to the standard radiotherapy group (5.1 months). The Nordic study showed a longer survival in patients over 70 years when treated with temozolomide or HFRT as compared to standard radiotherapy [12]. Based on these studies, it appears that HFRT is at least non-inferior to standard radiotherapy in elderly patients. A small phase III prospective study on elderly and/or frail in which patients were randomized to either a very short-course RT (25 Gy in five fractions delivered over 1 week) or commonly used HFRT (40 Gy in 15 fractions delivered over 3 weeks) [51], and showed an overall survival of 7.9 months in the 1 week radiotherapy group and 6.4 months in the 3 weeks radiotherapy group. However, the 1-week short course radiotherapy for GBM is controversial, and not commonly utilized in mainstream practice.

#### **11. Chemotherapy**

The alkylating agent temozolomide is the drug of choice for glioblastoma. Traditional treatment protocol that combines temozolomide (75 mg/m2 /day) with standard RT/HFRT after surgical resection followed by 200 mg/m<sup>2</sup> for 5 days with cycles repeated every 28 days for up to six cycles is the choice for patients with a good KPS (≥70) [21, 76]. Elderly glioblastoma patients who have a good KPS and reasonably controlled co-morbidities can be treated with the standard combined chemoradiation and adjuvant chemotherapy [7, 77]. No prospective study on elderly patients so far has compared standard chemoradiotherapy with hypofractionated chemoradiotherapy.

However, for fragile elderly patients who cannot tolerate the standard treatment, the use of temozolomide alone is non-inferior to standard radiation alone. A phase III randomized trial in patients older than 65 tested with dense temozolomide regimen to 6 weeks standard radiotherapy and found that temozolomide alone was non-inferior to radiotherapy alone [50]. This study also confirmed the role of MGMT as a predictive biomarker for chemotherapy monotherapy in these patients. The most common side effects described in this population are fatigue and thrombocytopenia [13]. Malmström et al., in the Nordic trial that included a arm of temozolomide found an overall survival (OS) significantly longer specially in patients above 70 years old with MGMT promoter methylated (9.7 vs. 6.8 months, compared to non- methylated). A phase III randomized trial of patients 65 years or older tested HFRT alone (40 Gy in 15 fractions) vs. HFRT concurrent with temozolomide, followed by adjuvant temozolomide [78]. The median overall survival was longer in the combined group than with radiotherapy alone (9.3 vs. 7.6 months). The benefit with combined treatment was much greater in the MGMT methylated GBMs. A non-significant survival benefit was also found in the MGMT unmethylated GBM patients. For MGMT unmethylated elderly GBM patients, RT only should be favored over temozolomide monotherapy [50].

Bevacizumab is not recommended for people with newly diagnosed glioblastoma due to high rates of adverse events and no improvement in overall survival [79–81]. In select cases, bevacizumab may be cautiously used to treat tumor-related edema and to avoid the side-effects of steroids.

#### **12. Tumor-treating field**

Tumor-treating field (TTF) is a device which is worn locally over the patient's shaved scalp and is FDA approved for treatment of glioblastoma. It delivers lowintensity alternating electric field to the tumor and thus has an anti-mitotic effect on glioblastoma. A phase III randomized clinical trial showed that the overall survival was significantly longer in the chemoradiotherapy + adjuvant temozolomide + TTF group (20.9 months) as compared to chemoradiotherapy + adjuvant temozolomide group (16 months) [7]. This study enrolled 695 patients with glioblastoma over the age of 18 and included 134 patients aged 65 or older. Patients 65 years or older had significantly increased survival on addition of TTF vs. temozolomide alone (HR, 0.51; 95% CI, 0.33–0.77). Device side-effects are mild as compared to chemotherapy or radiation and usually consist of mild to moderate skin toxicity underneath the transducer arrays. TTF is a local therapy and needs to be worn daily for long-term, optimally for over 18 h a day. Elderly patients will often require lifestyle modification and caregiver support when using it. It can be an attractive antineoplastic therapy for elderly as it does not have systemic side-effects.

#### **13. Conclusion**

GBM is the GBM is the most common primary brain tumor in the elderly population. It has been shown that GBM has a unique more aggressive biology in aged patients. Molecular patterns have not been thoroughly elucidated yet and many of these factors are thought to have a negative impact to the prognosis in these patients. If well-tolerated, surgical treatment should aim at gross total resection, and comprehensive pre-operative assessment is recommended. An active post-operative care can reduce the length of stay in these patients and consequently, the risk of post-operative complications and the incidence of delirium. Selected elderly patients with good performance status and well-controlled co-morbidities may receive standard 6 weeks of combined chemoradiotherapy, adjuvant temozolomide and TTF or HFRT combined with temozolomide. In patients with unmethylated tumors and poor KPS patients, HFRT alone has been commonly indicated. Chemotherapy alone is an option for patients with a low performance status and whose tumor is hypermethylated. Elderly patients with GBM represent a special and vulnerable group. Treatment in the elderly and very elderly patients with glioblastoma requires an individualized plan with a multi-disciplinary team. Patient's age, KPS, MGMT status, patient's wishes, and even social factors should guide the overall treatment plan.

*Glioblastoma - Current Evidence*

#### **Author details**

Raphael Bastianon Santiago1 , Hamid Borghei-Razavi1 \*, Mauricio Mandel1 , Bhavika Gupta2 , Asad Ali3 , Badih Adada1 and Surabhi Ranjan1

1 Department of Neurosurgery, Cleveland Clinic Florida, United States

2 School of Medicine, St George's University, Grenada

3 Dr. Kiran C. Patel College of College of Osteopathic Medicine, NOVA Southeastern University, United States

\*Address all correspondence to: borgheh2@ccf.org

© 2022 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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#### *Glioblastoma in Elderly Population DOI: http://dx.doi.org/10.5772/intechopen.106408*

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#### **Chapter 2**

## Glioblastomas: Molecular Diagnosis and Pathology

*Frank Y. Shan, Dachun Zhao, Carlos A. Tirado, Ekokobe Fonkem, Yi-lu Zhang, Dong-xia Feng and Jason H. Huang*

#### **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.

**Keywords:** epigenetic, biomarker

#### **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 oligodendrocyteoriginal 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.

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].

#### **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).*

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.

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.

**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.*

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].

*Glioblastomas: Molecular Diagnosis and Pathology DOI: http://dx.doi.org/10.5772/intechopen.105472*

#### **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 highgrade 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 [10–14]. 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 melanomalike 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 welldeveloped intensely staining network around spindle cells, and the glial component is seen as reticulin-free nests, which are immunoreactive for GFAP (**Figure 4B**) [24].

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 *Glioblastomas: Molecular Diagnosis and Pathology DOI: http://dx.doi.org/10.5772/intechopen.105472*

#### **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).*

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,

*Glioblastomas: Molecular Diagnosis and Pathology DOI: http://dx.doi.org/10.5772/intechopen.105472*

#### **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).*

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.

#### **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).

**Figure 7.** *1p/19q codeletion by FISH (A, 1p deletion red; B, 19q deletion red).*

#### *Glioblastoma - Current Evidence*

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 IDHmutant 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:


*Glioblastomas: Molecular Diagnosis and Pathology DOI: http://dx.doi.org/10.5772/intechopen.105472*

• 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.

#### **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.

*Glioblastomas: Molecular Diagnosis and Pathology DOI: http://dx.doi.org/10.5772/intechopen.105472*

*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 (posttranslational 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,


**Table 1.**

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

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 ligationdependent probe amplification (MS-MLPA) [31, 34].

Key points:


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**).

#### **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 *Glioblastomas: Molecular Diagnosis and Pathology DOI: http://dx.doi.org/10.5772/intechopen.105472*

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**


*Glioblastoma - Current Evidence*

#### **Author details**

Frank Y. Shan1,2\*, Dachun Zhao3 , Carlos A. Tirado1 , Ekokobe Fonkem<sup>2</sup> , Yi-lu Zhang2 , Dong-xia Feng2 and Jason H. Huang2

1 Department of Anatomic Pathology, Texas A&M University, Baylor Scott and White Health, College of Medicine, Temple, TX, USA

2 Department of Neurosurgery, Texas A&M University, Baylor Scott and White Health, College of Medicine, Temple, TX, USA

3 Department of Pathology, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, DongCheng District, Beijing, P.R. China

\*Address all correspondence to: yshan918@gmail.com

© 2022 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

*Glioblastomas: Molecular Diagnosis and Pathology DOI: http://dx.doi.org/10.5772/intechopen.105472*

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#### **Chapter 3**

## Perspective Chapter: Glioblastoma of the Corpus Callosum

*Daulat Singh Kunwar, Ved Prakash Maurya, Balachandar Deivasigamani, Rakesh Mishra and Amit Agrawal*

#### **Abstract**

Glioma is the most common malignant tumour of the brain, in which glioblastoma (GBM) is the most aggressive form which infiltrates through the white fibre tracts. Corpus callosum (CC) is most invaded by GBM, it carries poor prognosis as mostly these tumours are not touched upon due to the belief of post operative cognitive decline, or there is incomplete resection leading to tumour recurrence. However current advancement in technology, operative techniques and better understanding of nature of CC-GBM, maximal safe resection is being carried out with better outcomes in comparison with the GBM without infiltration of CC.

**Keywords:** butterfly glioma, butterfly glioblastoma, corpus callosum, glioma, glioblastoma, surgical resection, survival

#### **1. Introduction**

Glioblastoma multiforme originates in the cerebral white matter, accounts for 12–15% of all intracranial neoplasms and is the most common primary intra-axial malignancies [1]. Corpus callosum is the largest interhemispheric commissure connecting two identical cortical areas, and it acts as a white matter bridge between two hemispheres for tumour cells to migrate [2]. These are often reported arising from frontal and parietal lobes. Butterfly gliomas involving the corpus callosum characteristically appear as "butterfly" on imaging as the tumour has contiguous extension through the corpus callosum into both the cerebral hemispheres [1, 3, 4]. The incidence of butterfly glioma ranges from 3 to 14% of all high-grade gliomas [5, 6], and the isolated corpus callosum GBM is a relatively unusual variant of butterfly glioblastoma and account for 3% of all GBM [7]. The butterfly GBM of the corpus callosum can be anterior involving genu or less commonly can be posterior involving splenium [1]. Involvement of the corpus callosum can be on one side or either side involving both cerebral hemispheres (butterfly GBM) [8, 9]. Involvement of the corpus callosum makes the resection difficult and carries a poorer prognosis [10]. In this chapter, we discuss the pathology, clinical and imaging characteristics of glioblastomas involving the corpus callosum and review the management and outcome of these subgroup of tumours.

### **2. Clinical features**

Glioblastoma of the corpus callosum is characterised by a rapidly progressive deteriorating clinical course [11]. Progressive tumour growth in CC causes mass effect and white matter network connectivity changes (due to oedema or direct infiltration) [12]. Because of its location corpus callosum, glioblastomas involve the highly eloquent area of the brain, leading to impaired higher mental function, severe neurological deterioration and features of raised intracranial pressure (headache, vomiting and altered sensorium) [11, 13]. The myriad of symptoms of corpus callosum involvement includes non-specific headaches, paresis, seizures, depression, mutism, ataxia, behavioural abnormalities and Cotard's syndrome [14–16]. Tumours involving the splenium can lead to memory and cognitive function as several associative pathways pass through this area making the outcome further poorer [17].

### **3. Imaging**

CT scan with contrast administration can be used as screening tool; however, post-contrast MRI is the investigation of choice for detail evaluation and

#### **Figure 1.**

*Axial T1WI with contrast showing lesion involving the corpus callosum (at the genu) with main bulk towards the left side and crossing the midline to invade the right frontal lobe. The red arrows indicate the pushed anterior cerebral arteries towards the right side due to mass effect.*

*Perspective Chapter: Glioblastoma of the Corpus Callosum DOI: http://dx.doi.org/10.5772/intechopen.110019*

management including surgical planning [7, 18, 19]. Typically, corpus callosal GBM appears as a butterfly-shaped lesion with heterogeneous enhancement with areas of necrosis and haemorrhages with irregular postcontrast peripheral enhancement (**Figure 1**) [7, 18]. Coronal as well as sagittal fluid-attenuated inversion recovery images shall help in delineating the lesion and their relationship with surrounding structures better, [18] and diffusion tensor imaging shall help for the identification of white fibre tracts [20]. Pre-operative planning of tumour removal based on connectomics (machine learning-based algorithm which incorporates DTI and important cerebral network) is also available now [21].

#### **4. Differential diagnosis**

A number of pathologies those involve corpus callosum can mimic butterfly glioblastomas including other lesser grade variants of gliomas involving corpus callosum, [22–25] lymphoma, metastasis, [26] toxoplasmosis, [27] demyelinating butterfly pseudo glioma, [28] and neuronal ceroid-lipofuscinosis (Kufs' disease) [29] because of its multiplanar capability, MRI with contrast enhancement and FLAIR sequence [7, 18] can help to differentiate these lesions from each other; however, in doubtful cases the biopsy shall help to make the diagnosis.

#### **5. Management**

The aim of management is to improve patient's functionality and quality of life by relieving the symptoms and minimising the complications. Even though there are advances in immunotherapy, targeted therapy and oncolytic viral therapy most patients with CC-GBM suffer from limited survival. Currently, maximal safe resection with adjuvant chemo-radiotherapy remains gold standard [30–32]. Recent advances in the management of brain tumours have made resection of the corpus callosum glioblastomas preferred, possible and safe [33, 34]. Surgery improves overall survival, and it is superior to biopsy [4, 35, 36]. Surgical approaches help in reducing the tumour burden [11, 35, 37, 38] and also provide tissue sample for pathologic and molecular characterisation of the tumour (IDH 1/2 mutation or MGMT promoter methylation or both), thus guiding the further adjuvant management approaches [35]. Surgical resection can also be facilitated by intraoperative magnetic resonance imaging MRI-guided laser interstitial thermal therapy (LITT) techniques as this will increase the efficacy and safety of the procedure [37, 39–41]. Evidence suggests that preoperative KPS score, adjuvant radio chemotherapy and extent of surgical resection (EoR) have impact on survival besides patient's age. In a systematic review done by Palmisciano *et al.* [12], they say that resection of glioma infiltrating the corpus callosum has no significant changes in the post operative complications. Gross total resection of the tumour increases overall survival. Foster *et al.* [42] say that many patients with glioma infiltrating the corpus callosum rarely undergo surgical removal in fear of the post op neuropsychological sequelae. Authors hypothesise that the neuropsychological deficits are mainly due to tumour. Removing tumour reduces the mass effect and improves the microenvironment of the surrounding neurons; this may improve the neurocognitive and neurological function. In a prospective analysis done by them in 21 patients, they found that the neurocognitive decline post operatively was present in 75% of patients who presented with a median KPS of 100%.

#### **Figure 2.**

*Intraoperative photograph of tumour resection with the use of sodium fluorescence dye. The blue arrow indicates the plane of tumour-brain interface which was obvious after sodium fluorescence dye administration and facilitated the tumour decompression.*

#### **Figure 3.**

*Representative sketch depicting the corpus callosum and related neuroanatomical structures encountered during surgical resection. The septal nuclei (under orange oval area) need to be preserved during tumour decompression.*

But surprisingly after 6 months a very few had impairment in attention, executive functioning, memory and depression. Authors strongly suggest that surgical resection of tumour might outweigh morbidity. Complications like motor deficits, cognitive decline post operatively is due to manipulation of the white fibres of CC and post operative edema (**Figure 2**) [36, 43].

Photo dynamic tumour visualisation technology is very helpful in achieving maximal extent of resection (i.e. supra marginal resection) which is the only modifiable factor linked with overall survival of the patients. Sodium fluorescein (**Figure 2**) and 5-Aminolevulinic acid (5-ALA) are the agents currently being used. In a recent study

#### **Figure 4.**

*Figure demonstrating white fibres through which tumour cells from one part of the brain reaching corpus callosum and travels to other side. The light green colour lesion is representing a lesion in the right frontal lobe infiltrating the forceps minor and traversing towards the opposite side.*

done on peritumoral region, they found that 5-ALA staining extends beyond the sodium fluorescein-stained areas, even then there are tumour positive cells beyond this region [44]. Combining both fluorescein sodium and 5-ALA gives very good background information of the glioma cells and is more effective in supra marginal resection [33, 45, 46] current understanding is that fluorescein and 5-ALA should be supplemented with supplemented with intra-operative neurophysiological monitoring for better clinical outcome as well as overall survival [44].

In cases of glioma infiltrating the genu and rostrum of the corpus callosum, one should be careful not to enter the subcallosal region (contains septal nucleus) during resection (**Figures 3** and **4**). As this may cause psychiatric disturbances along with cognitive decline, this has been pointed out by Sughrue *et al.* [34].

However, because of its unique location and spread, in comparison with other GBMs, the conservative resection of corpus callosum is possible, thus reducing the chances of overall survival [9–12]. Temozolomide alone or in combination has been shown as a safer alternative in elderly population [26, 28, 42, 43].

#### **6. Outcome**

In spite of advances in maximal safe surgical resection techniques, availability of adjuvant radiotherapy and temozolomide chemotherapy, as for other glioblastomas the prognosis in cases of corpus callosal glioblastomas is dismal [3, 4, 19, 25, 35, 39, 47]. In literature, the overall survival in cases of butterfly glioblastomas is in weeks to months, and the median survival of 3 months and a six-month survival is only 38% [3, 19, 22, 24]. Median overall survival of a CC infiltrated glioblastomas is 10.7 months, whereas it is 13.2 months in a non-CC infiltrated glioblastoma [36]. In a series of 215 patients where

the corpus callosum was involved, overall survival was less than <6 months [48]. It is also observed that there are higher rates of recurrence in whom the infiltrated part of tumour in corpus callosum was not removed [36, 49]. However, their isolated case of long-term survival, in a report the patient survived the disease for 5 years and 2 months after the initial diagnosis [50].

### **7. Conclusion**

Glioblastoma infiltrating the corpus callosum is rare yet highly invasive. With the improved intra-operative adjuncts, surgical techniques and concepts, there is higher tumour resection rates with minimal complications. While managing corpus callosal tumours, one should always aim for safe maximal resection with multimodal approach if the situation permits. However, in spite of the advances in the diagnosis and management techniques, there is not much improvement in the overall outcome of these patients.

### **Author details**

Daulat Singh Kunwar1 \*, Ved Prakash Maurya<sup>2</sup> , Balachandar Deivasigamani3 , Rakesh Mishra4 and Amit Agrawal5

1 Department of Radiotherapy and Clinical Oncology, Government Doon Medical College, Dehradun, Uttarakhand, India

2 Department of Neurosurgery, Sanjay Gandhi Post Graduate Institute of Medical Sciences, Lucknow, India

3 Department of Neurosurgery, Apollo Cancer Centre, Chennai, India

4 Department of Neurosurgery, Institute of Medical Sciences, Trauma Centre and Mahamana Centenary Superspeciality Hospital, Banaras Hindu University, Varanasi, India

5 Department of Neurosurgery, All India Institute of Medical Sciences, Bhopal, Madhya Pradesh, India

\*Address all correspondence to: daulatsinghk@yahoo.com

© 2023 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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#### **Chapter 4**

## Canine Glioma as a Model for Human Glioblastoma

*Nicole M. Yost and James M. Angelastro*

#### **Abstract**

Glioblastoma, a high-grade diffuse glioma, carries a poor clinical prognosis despite decades of extensive research on the genetic and molecular features of disease and investigation of experimental therapeutics. Because spontaneous canine glioma and human glioblastoma share many clinicopathologic characteristics, recent efforts have focused on utilizing companion dogs as a preclinical model for both research and therapeutic development. A detailed investigation of the canine disease, with particular attention to the genetic and molecular profile, is important in order to allow translation of specific clinical findings from canines to humans and vice versa. In this chapter, we investigate the most common genetic, molecular, and epigenetic alterations associated with canine and human glioma. Appropriate implementation of the canine glioma model may provide valuable information to improve both human and veterinary patient care.

**Keywords:** glioma, glioblastoma, canine, spontaneous model, translational neuro-oncology, comparative biology, genetics, molecular pathology

#### **1. Introduction**

Gliomas are the most common type of malignant primary central nervous system (CNS) neoplasm in humans within the United States [1]. Glioblastoma (GBM), a World Health Organization (WHO) grade IV glioma, is a particularly aggressive tumor, and it accounts for nearly half of the malignant CNS tumors in humans, with an average incidence of over 12,000 cases each year [1, 2]. Even with intensive therapy involving surgery, radiation therapy, chemotherapy, and the most recent FDA-approved therapy utilizing antimitotic alternating electrical fields, the median survival time for patients with GBM is less than 2 years [3].

Companion dogs also spontaneously develop gliomas, including high grade variants that are similar to human glioma and glioblastoma [4]. These canine gliomas share many clinicopathologic features with human disease, such as comparable imaging characteristics, genetic and molecular aberrations, tumor microenvironments, and histopathologic characteristics [5–9]. Correspondingly, the Comparative Oncology Program within the National Cancer Institute developed a Comparative Brain Tumor Consortium (CBTC) to further investigate and utilize spontaneously arising brain tumors in dogs as a model of the human disease with specific emphasis

on comparative glioma [10]. As such, the spontaneous canine glioma model has gained attraction as a preclinical tool to improve the success of human clinical trials by bridging the gap between laboratory models of glioma and human patients.

Subsequent large-scale studies have greatly improved our diagnostic classification and molecular understanding of canine gliomas and have allowed more direct comparisons to human glioma and glioblastoma [11, 12]. While many similarities continue to exist between canine and human glioma, it is also important to characterize the differences between the canine and human disease to ensure that the model is utilized effectively and appropriately. Further investigation into canine glioma, with a focus on comparative molecular and genetic characteristics, can help establish which novel therapeutics can best harness the canine spontaneous glioma model and allow maximal possible benefit to both human and animal patients with gliomas.

#### **2. Overview of canine glioma**

Gliomas are the second most common primary intracranial tumor among dogs [4, 13] and have an overall prevalence of 0.9% in the canine population [4]. Gliomas tend to occur in adult dogs, with a median age of diagnosis of approximately 8 years and an increasing prevalence with increasing age [4, 14]. No significant difference in the frequency of intracranial tumors in male versus female dogs have been shown [13], although several recent studies have documented a slightly higher rate of diagnosis in males [14, 15]. Brachycephalic dog breeds, including Boston Terriers, French Bulldogs, English Bulldogs, Boxers, and English Toy Spaniels, are at significantly higher risk of developing gliomas [4] and are overrepresented, collectively comprising 78% of all cases of canine glioma [14]. A recent genome-wide association study identified a genetic locus and 3 candidate genes that are linked to glioma susceptibility in dogs and may have been under selection among brachycephalic breeds [16].

Common clinical signs among dogs with gliomas include: seizure, gait abnormalities, and mentation and behavior changes [14]. Seizures are particularly common among dogs with a specific type of glioma called oligodendroglioma, and these patients are 3 times more likely to experience seizures than dogs with any other type of primary CNS tumor [13]. Cerebrospinal fluid analysis results are variable among dogs with primary brain tumors, as both inflammatory profiles and normal protein and cell counts have been documented in canine gliomas [13, 14]. Although extracranial metastasis of primary gliomas has not been reported in thoracic and abdominal imaging nor post-mortem analysis at necropsy [13, 14], other unrelated concurrent neoplastic processes have been identified both antemortem and at necropsy in canine glioma patients [13].

Computed tomography (CT) and magnetic resonance imaging (MRI) are the two most widely used imaging modalities to aid in the diagnosis and assessment of canine gliomas. MRI is generally considered the preferred modality for identification of intracranial disease, although CT has been shown to detect mass lesions within the brain in 90% of primary brain tumor cases [13] and has similar ability to measure tumor margins as MRI [17]. On MRI, canine gliomas are generally hypointense on T1-weighted images (T1WI) and hyperintense on T2-weighted images (T2WI) [7]; however some reports note that canine gliomas on T1WI and T2WI are also commonly isointense and of mixed intensity [18]. Generally, low grade canine gliomas have lower levels of contrast enhancement, are less commonly associated with cystic structures, and are located more superficially than high grade gliomas [19]. Overall, MRI

#### *Canine Glioma as a Model for Human Glioblastoma DOI: http://dx.doi.org/10.5772/intechopen.106464*

is relatively sensitive (approximately 90%) at identifying canine intracranial tumors [18]; however, both MRI and CT are inaccurate predictors of canine glioma type and grade, and ultimately biopsy with histopathology is required for diagnosis [17].

The histopathologic classification scheme of gliomas in both humans and dogs has undergone significant changes in the past several years [2, 11], but generally, gliomas are defined as tumors that resemble glial cells histologically [20]. The two most common types of gliomas are oligodendrogliomas and astrocytomas, and in humans, these gliomas are graded from a scale of grade I to IV based on increasing characteristics of malignancy, as defined by WHO [20, 21]. Molecular and genetic characteristics have been incorporated into the human glioma grading scheme and are expected to be added to the recently revised canine histopathologic glioma classification system [2, 11]. Currently, the three types of gliomas in dogs are oligodendroglioma, astrocytoma, and undefined glioma. These subtypes are then classified as either low- or high-grade based on factors such as necrosis, microvascular proliferation, amount of mitotic activity, and cellular features of malignancy [11].

One important difference between the human and canine disease is the relative frequency of glioma subtypes among patients. The vast majority of human gliomas (approximately 78%) are astrocytic tumors, with 58% of those being the highly malignant GBM [1]. A recent necropsy report utilizing the updated canine glioma classification system reports that astrocytomas may make up as low as 19% of all canine gliomas, with the majority of astrocytomas and oligodendrogliomas being high grade (94% and 84%, respectively) [14]. However, the percentage of canine glioma samples diagnosed as astrocytoma is variable within the literature, with some necropsy reports noting that 35% and 60% of all canine gliomas are astrocytic tumors [4, 13].

Similar treatments options exist for canine glioma, including surgery, radiation therapy, and chemotherapy [15, 22, 23]. In a systematic review of treatment modalities in canine brain tumors, the median survival time of dogs with suspected intracranial gliomas is reported as 226 days [24]. However, euthanasia is also commonly elected for companion dogs diagnosed with gliomas, and one study found that nearly half of all dogs with glioma were euthanized on the day of diagnosis [14]. As such, canine spontaneous glioma is a disease that is associated with significant morbidity and mortality, and novel treatments to improve survival times are clearly still needed.

#### **3. Comparative genetic and molecular signature**

Our understanding of the molecular aberrations associated with gliomas has dramatically expanded over the last several decades. In humans, it was found that specific genetic and molecular characteristics are closely linked to glioma biologic behavior and prognosis [20]. Thus, the WHO CNS tumor classification criteria began to incorporate molecular parameters in addition to classic histopathological characteristics into the glioma grading scheme in the 2016 update [2]. In alignment with the goal to utilize canine glioma patients as a model of the human disease, the CBTC assembled a Glioma Pathology Board to revise the canine glioma classification system in a way such that genomic data can be incorporated, mirroring the human classification system [11].

In order to assess the extent to which the spontaneous canine glioma model can be utilized as a model of the human disease, a detailed investigation of what is known about the genetic landscape of canine gliomas is warranted. Genetic alterations that are commonly encountered in human glioblastoma and canine glioma will be discussed, including dysregulation of the receptor tyrosine kinase (RTK)/Ras/

phosphoinositide 3-kinase (PI3K) pathway, the p53 pathway, and the retinoblastoma (Rb) pathway, as well as other specific genes, proteins, and epigenetic factors involved in canine and human glioma. See **Table 1** for a list of abbreviations used for oncogenes and tumor suppressor genes discussed. See **Table 2** for a summary of the comparative somatic mutation rates among common glioma drivers in humans and dogs.

#### **3.1 RTK/Ras/PI3K pathway**

Tyrosine kinase receptors are commonly altered in human glioblastoma. Brennan et al. found that at least one RTK is either amplified or mutated in 67% of


#### **Table 1.**

*Abbreviations of oncogenes and tumor suppressor genes discussed.*


#### **Table 2.**

*Somatic mutation rates of selected genes commonly altered in canine glioma and human glioblastoma.*

human GBM cases [25]. The most frequently mutated RTK in human GBM is *EGFR* (epidermal growth factor receptor) with a somatic mutation rate of 26%, followed by *PDGFRA* (platelet-derived growth factor receptor A) with a somatic rate of 4% [25]. Both genetic alterations have also been documented in canine glioma; however, the relative frequency is reversed, with a somatic mutation rate of 4% and 21% for *EGFR* and *PDGFRA*, respectively [12]. Utilizing estimates of clonal driver mutations within gliomas, Amin et al. found that clonal *PDGFRA* and *EGFR* mutations occur early on during gliomagenesis within both human and canine gliomas, suggesting molecular similarity among canine and human glioma [12].

*EGFR* gene amplification is rarely identified in canine glioma, with one report documenting *EGFR* amplification in 3% of cases [8], but overexpression of EGFR protein among dogs with glioma is common. Approximately half of all dogs with gliomas have been reported to have overexpression of EGFR, with significantly greater expression levels among high grade compared to low grade gliomas [26]. Although EGFR mRNA overexpression is seen consistently across both canine astrocytomas and oligodendrogliomas [27], EGFR protein overexpression tends to be more common among astrocytomas and more rarely identified in canine oligodendrogliomas [28].

The *PDGFRA* K385I/M mutation found in a subset of canine gliomas is one of the drivers of glioma in dogs [12]. *PDGFRA* gene amplification is present in nearly half of all canine glioma cases and is particularly common in oligodendrogliomas due to a large gain on canine chromosome 13 [8]. One study found overexpression of PDGFRA mRNA among all canine oligodendrogliomas and nearly half of canine astrocytomas [27]. PDGFRA protein expression patterns are similar, with the highest frequency of PDGFRA overexpression among high grade oligodendrogliomas and fewer numbers of samples overexpressing PDGFRA among canine astrocytomas. Canine astrocytoma PDGFRA overexpression frequency decreases in parallel with decreasing tumor grade [28].

Although genetic alterations in other tyrosine kinase receptors are less common than in EGFR and PDGFRA, many of these receptors have also been investigated as potential targets for glioma therapeutics [29], and will thus be briefly discussed. VEGFR (vascular endothelial growth factor receptor)-1 and VEGFR-2 mRNA overexpression is present in nearly all canine gliomas, with significantly increasing expression correlating with increasing astrocytoma grade [27]. Amplification or mutations involving *FGFR* (fibroblast growth factor receptor) is uncommon in human glioblastoma, with an alteration rate of 3.2% [25], and while the somatic mutation rate for canine gliomas is similarly low, around 1–2% [12], the frequency of *FGFR-1* amplification in canine glioma is notably higher, around 30% [8].

Downstream signaling molecules in the RTK/Ras/PI3K pathway also play important roles in both human and canine glioma and will be investigated further in this section. Somatic mutations involving the tumor suppressor gene *NF1* (neurofibromin 1) occur with similar frequency in human and canine glioma, at a rate of about 11% and 7%, respectively [12, 25]. *NF1* frameshift mutations tend to be late events in the development of gliomas in both humans and dogs [12]. Homozygous losses of *NF1* are uncommon in canine gliomas, being present in about 3% of cases [8]. In a study investigating oligodendrogliomas in brachycephalic breeds, NF1 was not differentially expressed in tumor cells and had similar to expression levels in normal tissue [30].

The tumor suppressor gene *PTEN* (phosphatase and tensin homolog) is the most frequently altered gene in human glioblastoma, with a somatic mutation rate of 31% [25]. Although somatic mutations have not been documented involving *PTEN* in canine gliomas, copy number losses are present in approximately 15% of canine gliomas [8]. With regards to PTEN protein expression, variable expression among

canine gliomas and normal CNS tissue has been observed, with a lack of differential expression in tumor tissue [5, 30].

The second most commonly encountered somatic mutation in canine glioma involves the gene *PIK3CA* (phosphatidylinositol 3-kinase catalytic subunit alpha), which is altered in 14% of cases [12]. *PIK3CA* is also mutated with similar frequency in human glioblastoma, with a somatic mutation rate of 11% [25]. The *PIK3CA* H1047R/L mutation found in a subset of canine gliomas is one of the drivers of glioma in dogs [12]. Mutations involving *PIK3CA* are characterized as early mutations driving tumor formation in canine and pediatric but not adult glioma [12]. However, a closely related gene, *PIK3R1* (phosphatidylinositol 3-kinase regulatory subunit 1), is more frequently altered in human glioblastoma than in canine glioma with somatic mutation rates of 11% and 1% [12, 25].

#### **3.2 p53 and Rb pathways**

*TP53* (tumor protein 53), a tumor suppressor gene, is one of the most frequently altered genes in human glioblastoma, with a somatic mutation rate of 29% [25]; however *TP53* is infrequently mutated in canine glioma, with a somatic mutation rate of only 5% [12]. Although *TP53* somatic mutations among dogs with glioma are rare, focal somatic copy number alterations are slightly more common, at a rate of 12% [12]. TP53 protein expression is most common in canine astrocytic tumors, with more variable and decreased expression among dogs with oligodendrogliomas [5]. TP53 mRNA expression is upregulated relative to normal tissue in brachycephalic breeds with oligodendrogliomas [30]. *CDKN2A* (cyclin-dependent kinase inhibitor 2A) deletions are commonly seen in human GBM, at a rate of 58% [25]. While *CDKN2A* deletions are also present in canine glioma, these mutations are only in astrocytomas and occur at a lower rate of approximately 12% [12]. Although *MDM2* (mouse double minute 2 homolog) amplifications in canine gliomas have not been documented, *MDM4* is amplified in 42% of canine gliomas [8]. Overall p53 pathway copy number alterations are present in 76% of canine gliomas [8], which is similar to the frequency of p53 pathway alterations in 85% of human glioblastomas [25].

*RB1* (retinoblastoma 1) somatic mutations are present in human GBM at a rate of 9% [25], while canine glioma *RB1* somatic mutations are much less common, with only 1% of samples affected [12]. Although *RB1* somatic mutations among dogs with glioma are rare, focal somatic copy number alterations are more common, at an overall rate of 21% [12]. *RB1* deletions are most common among canine oligoastrocytomas, followed by oligodendrogliomas and astrocytomas, with gene losses occurring in 80%, 60%, and 27% of samples, respectively [8]. RB1 protein levels in canine glioma are overexpressed, and most of the RB1 protein is dephosphorylated [5]. *CDK4* (cyclin-dependent kinase 4) is not amplified in canine glioma, and *CDK6* is only amplified in 3% of canine glioma samples [8]. Overall Rb pathway copy number alterations are present in 79% of canine gliomas [8], which exactly mirrors the rate (79%) at which human glioblastomas contain Rb pathway alterations [25].

#### **3.3 Other genetic and epigenetic alterations involved in canine and human Glioma**

The classic *IDH1* (isocitrate dehydrogenase 1) R132H mutation commonly seen in human low grade gliomas and secondary recurring human GBM [31] has not been observed in canine glioma [32, 33]. However, mutations involving *IDH1* are found infrequently in canine gliomas, with a mutation rate of 4%, and the *IDH1*

*Canine Glioma as a Model for Human Glioblastoma DOI: http://dx.doi.org/10.5772/intechopen.106464*

R132C mutation found in a small subset of canine gliomas is one of the drivers of glioma in dogs [12].

The transcription factor ATF5 (activating transcription factor 5), has been shown to be overexpressed in several types of cancers in humans [34, 35], and ATF5 mRNA and protein are overexpressed in human low grade astrocytoma and GBM, with the highest expression in GBMs [36]. ATF5 protein expression is also elevated in canine gliomas, with the highest levels of expression in canine GBM [37].

Canine glioma is reportedly more similar to human pediatric glioma than adult glioma with respect to several different factors. Both canine and human pediatric glioma cases contain at least 1 significantly mutated gene in approximately half of the cases; this is contrasted with adult human gliomas, which carry at least 1 significantly mutated gene over 90% of the time [12]. Although canine glioma has a relatively low mutational burden, aneuploidy (characterized by arm-level copy gains) is common in canine gliomas. The median percent of the canine genome affected by copy number alterations in canine glioma is 25%, which is similar to human pediatric glioma (19–26% of the genome); both of which were higher than adult glioma (8–18% of the genome) [12]. The DNA methylation pattern of canine gliomas was found to be characterized as pediatric glioma in 78% of samples analyzed, with the other remaining 13% and 9% of cases being classified as IDH wild-type adult and IDH-mutant adult glioma, respectively [12].

#### **4. Conclusion**

Both human glioblastoma and canine glioma are diseases that carry a grim prognosis for patients. Because dogs develop gliomas spontaneously and with similar frequencies and clinicopathologic features of disease, canine glioma has recently been proposed as a preclinical model for both research efforts and novel therapeutic development prior to clinical trials in humans. In order to best utilize this model, a thorough investigation into what is currently known about canine glioma is of paramount importance.

While many similarities exist between human and canine glioma, several key differences are essential to document so that this model can be used appropriately. The key differences between human and canine glioma that are highlighted in this review include: the relative frequency of glioma histologic subtypes, the frequency of specific genetic variants among drivers of glioma formation, the overall genomic mutational burden, the relative frequency of aneuploidy, and the pattern of DNA methylation. With regards to aneuploidy and epigenetic changes, canine glioma appears to be more similar to pediatric than adult glioma.

These differences are particularly important to consider with respect to investigational therapeutics. New drugs and other therapies that specifically target or otherwise harness these features of glioma to treat the disease may yield different results among canines and humans with gliomas. Additionally, canine glioma may serve as a more reliable model for human pediatric glioma in certain genetic and epigenetic studies.

#### **Acknowledgements**

Funding was provided by the Students Training in Advanced Research (STAR) Program through a National Institutes of Health T35 grant (OD010956).

### **Conflict of interest**

J.M. Angelastro was on the scientific advisory board member of Sapience Therapeutics (2016–2020), which has licensed the ATF5 technology to treat one of the cancers, glioblastoma, from Columbia University, and is co-inventor on patents owned by Columbia University (New York, NY) and patents owned by Columbia University and the University of California, Davis (Davis, CA).

#### **Author details**

Nicole M. Yost1,2 and James M. Angelastro3 \*

1 School of Veterinary Medicine, University of California, Davis, CA, USA

2 Center for Comparative Medicine, Baylor College of Medicine, Houston, TX, USA

3 Department of Molecular Biosciences, School of Veterinary Medicine, University of California, Davis, CA, USA

\*Address all correspondence to: jmangelastro@ucdavis.edu

© 2022 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

*Canine Glioma as a Model for Human Glioblastoma DOI: http://dx.doi.org/10.5772/intechopen.106464*

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[4] Song RB, Vite CH, Bradley CW, Cross JR. Postmortem evaluation of 435 cases of intracranial neoplasia in dogs and relationship of neoplasm with breed, age, and body weight. Journal of Veterinary Internal Medicine. 2013;**27**(5):1143-1152

[5] Boudreau CE, York D, Higgins RJ, LeCouteur RA, Dickinson PJ. Molecular signalling pathways in canine gliomas. Veterinary and Comparative Oncology. 2017;**15**(1):133-150

[6] Candolfi M, Curtin JF, Nichols WS, Muhammad AKMG, King GD, Pluhar GE, et al. Intracranial glioblastoma models in preclinical neuro-oncology: Neuropathological characterization and tumor progression. Journal of Neuro-Oncology. 2007;**85**(2):133-148

[7] Young BD, Levine JM, Porter BF, Chen-Allen AV, Rossmeisl JH, Platt SR, et al. Magnetic resonance imaging features of intracranial astrocytomas and oligodendrogliomas in dogs. Veterinary Radiology & Ultrasound. 2011;**52**(2):132-141

[8] Dickinson PJ, York D, Higgins RJ, Le Couteur RA, Joshi N, Bannasch D. Chromosomal aberrations in canine gliomas define candidate genes and common pathways in dogs and humans. Journal of Neuropathology and Experimental Neurology. 2016;**75**(7):700-710

[9] Lipsitz D, Higgins RJ, Kortz GD, Dickinson PJ, Bollen AW, Naydan DK, et al. Glioblastoma multiforme: Clinical findings. Magnetic Resonance Imaging, and Pathology in Five Dogs. 2003;**40**(6):659-669

[10] Leblanc AK, Mazcko C, Brown DE, Koehler JW, Miller AD, Miller CR, et al. Creation of an NCI comparative brain tumor consortium: informing the translation of new knowledge from canine to human brain tumor patients. Neuro-Oncology. 2016;**18**(9):1209-1218

[11] Koehler JW, Miller AD, Miller CR, Porter B, Aldape K, Beck J, et al. A revised diagnostic classification of canine glioma: Towards validation of the canine glioma patient as a naturally occurring preclinical model for human glioma. Journal of Neuropathology and Experimental Neurology. 2018;**77**(11):1039-1054

[12] Amin SB, Anderson KJ, Boudreau CE, Martinez-Ledesma E, Kocakavuk E, Johnson KC, et al. Comparative molecular life history of spontaneous canine and human gliomas. Cancer Cell. 2020;**37**(2):243-257.e7

[13] Snyder JM, Shofer FS, Van Winkle TJ, Massicotte C. Canine intracranial primary neoplasia: 173 cases (1986-2003). Journal of Veterinary Internal Medicine. 2006;**20**(3):669-675

[14] José-López R, Gutierrez-Quintana R, de la Fuente C, Manzanilla EG, Suñol A, Pi Castro D, et al. Clinical features, diagnosis, and survival analysis of dogs with glioma. Journal of Veterinary Internal Medicine. 2021;**35**(4):1902-1917

[15] Hubbard ME, Arnold S, Bin Zahid A, McPheeters M, Gerard O'Sullivan M, Tabaran AF, et al. Naturally occurring canine glioma as a model for novel therapeutics. Cancer Investigation. 2018;**36**(8):415-423. DOI: 10.1080/07357907.2018.1514622

[16] Truvé K, Dickinson P, Xiong A, York D, Jayashankar K, Pielberg G, et al. Utilizing the dog genome in the search for novel candidate genes involved in glioma development—genome wide association mapping followed by targeted massive parallel sequencing identifies a strongly associated locus. PLoS Genetics. 2016;**12**(5):1-22

[17] Stadler KL, Ruth JD, Pancotto TE, Werre SR, Rossmeisl JH. Computed tomography and magnetic resonance imaging are equivalent in mensuration and similarly inaccurate in grade and type predictability of canine intracranial gliomas. Frontiers in Veterinary Science. 2017;**4**(SEP):1-7

[18] Ródenas S, Pumarola M, Gaitero L, Zamora À, Añor S. Magnetic resonance imaging findings in 40 dogs with histologically confirmed intracranial tumours. Veterinary Journal. 2011;**187**(1):85-91

[19] Bentley RT, Ober CP, Anderson KL, Feeney DA, Naughton JF, Ohlfest JR, et al. Canine intracranial gliomas:

Relationship between magnetic resonance imaging criteria and tumor type and grade. Veterinary Journal. 2013;**198**(2):463-471. DOI: 10.1016/j. tvjl.2013.08.015

[20] Chen R, Smith-Cohn M, Cohen AL, Colman H. Glioma subclassifications and their clinical significance. Neurotherapeutics. 2017;**14**(2):284-297

[21] Louis DN, Ohgaki H, Wiestler OD, Cavenee WK, Burger PC, Jouvet A, et al. The 2007 WHO classification of tumours of the central nervous system. Acta Neuropathologica. 2007;**114**(2):97-109

[22] Magalhães TR, Benoît J, Néčová S, North S, Queiroga FL. Outcome after radiation therapy in canine intracranial meningiomas or gliomas. In Vivo (Brooklyn). 2021;**35**(2):1117-1123

[23] Moirano SJ, Dewey CW, Wright KZ, Cohen PW. Survival times in dogs with presumptive intracranial gliomas treated with oral lomustine: A comparative retrospective study (2008-2017). Veterinary and Comparative Oncology. 2018;**16**(4):459-466

[24] Hu H, Barker A, Harcourt-Brown T, Jeffery N. Systematic review of brain tumor treatment in dogs. Journal of Veterinary Internal Medicine. 2015;**29**(6):1456-1463

[25] Brennan CW, Verhaak RGW, McKenna A, Campos B, Noushmehr H, Salama SR, et al. The somatic genomic landscape of glioblastoma. Cell. 2013;**155**(2):462

[26] Fraser AR, Bacci B, le Chevoir MA, Long SN. Epidermal growth factor receptor and Ki-67 expression in canine gliomas. Veterinary Pathology. 2016;**53**(6):1131-1137

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Kass PH, et al. Expression of receptor tyrosine kinases VEGFR-1 (FLT-1), VEGFR-2 (KDR), EGFR-1, PDGFRa and c-Met in canine primary brain tumours. Veterinary and Comparative Oncology. 2006;**4**(3):132-140

[28] Higgins RJ, Dickinson PJ, Lecouteur RA, Bollen AW, Wang H, Wang H, et al. Spontaneous canine gliomas: Overexpression of EGFR, PDGFRα and IGFBP2 demonstrated by tissue microarray immunophenotyping. Journal of Neuro-Oncology. 2010;**98**(1):49-55

[29] Pearson JRD, Regad T. Targeting cellular pathways in glioblastoma multiforme. Signal Transduction and Targeted Therapy. 2017;**2**(June):1-11. DOI: 10.1038/sigtrans.2017.40

[30] Filley A, Henriquez M, Bhowmik T, Tewari BN, Rao X, Wan J, et al. Immunologic and gene expression profiles of spontaneous canine oligodendrogliomas. Journal of Neuro-Oncology. 2018;**137**(3):469-479. DOI: 10.1007/s11060-018-2753-4

[31] Cohen AL, Holmen SL, Colman H. IDH1 and IDH2 mutations in gliomas. Current Neurology and Neuroscience Reports. 2013;**13**(5):1-7

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**Chapter 5**

## Systemic Treatment in Glioblastoma

*María Ángeles Vaz, Sonia Del Barco Berrón, Raquel Luque, María Villamayor, Juan Manuel Sepúlveda Sánchez and María Vieito*

#### **Abstract**

Glioblastoma is the most common primary brain tumor and the initial treatment with maximal safe resection is not curative. In order to improve the prognosis, surgery is completed with radiotherapy and temozolomide, an oral chemotherapy, but overall survival remains poor. Therefore, new efforts are needed to improve these results. In fact, different systemic treatments have been tested but, nevertheless, few advances have been reached despite the development of large clinical trials. This chapter will review the most important findings, achievements, and main studies in this pathology. Standard of care in newly diagnosed and recurrent glioblastoma will be reassessed with the results of clinical trials with targeted agents and immunotherapy. Ongoing studies are evaluating advanced treatments, with chimeric antigen receptor T-cells, biospecific T-cell antibodies, tumor vaccines, and oncolytic viruses, although results are pending, a wide review of these new-generation agents is important to better understand the advances in glioblastoma in the coming years.

**Keywords:** glioblastoma, chemotherapy, immunotherapy, clinical trials

#### **1. Introduction**

Glioblastoma is the most frequent primary brain tumor in adults.

The median age of diagnosis is 64 years and the average age-adjusted incidence rate is 3.2 per 100.000 population [1].

Initial treatment includes maximal safe resection, radiotherapy, and chemotherapy with temozolomide based on a phase III pivotal study published in 2005.

Despite all of this, the prognosis remains poor with a median overall survival of 14 months [2].

Therefore, new efforts are needed to improve these results.

Systemic treatments have been tested in different clinical trials. Nevertheless, few advances have been reached.

This chapter will review the most important achievements and main studies in this pathology.

To understand the difficulties to advance in glioblastomas, here we expose some characteristics of this tumor.

Glioblastoma is characterized by the presence of several mechanisms of resistance to different treatments.

#### *Glioblastoma - Current Evidence*

One of them is the presence of the blood-brain barrier (BBB*).* The BBB is composed of a neurovascular structure, with specialized capillary endothelial cells adhered with tight junctions, a basal lamina, and a complex of astrocytic endfeet, pericytes, and intermittent end of neurons. Only small molecules can passively diffuse across this barrier. Other molecules need mechanisms such as pinocytosis or receptor or carried-mediated transcytosis.

Moreover, several drug-resistance proteins (such as P-glycoprotein and multidrug resistance-1) are expressed in the vessel wall to reinforce this barrier.

In glioblastoma, the BBB is heterogeneously disrupted with reduced tight junctions, altered pericytes, and astrocytic end-feet, leading to tumoral areas with different blood permeability to the different drugs [3].

Another mechanism of resistance is tumor heterogeneity, which is perhaps the most challenging obstacle to finding successful treatments for glioblastoma. At a cellular level, glioblastoma tumors are composed of various groups of cells and glioma stem cells (GSCs), each with a specific transcriptional signature.

Moreover, glioblastoma is also characterized by spatial heterogeneity due to the presence of diverse hypoxia gradients and heterogeneity of the tumoral microenvironment.

On the other hand, primary and recurrent glioblastoma can have subclonal genetic alterations, with the presence of regions with different drug sensitivity [3].

Other studied factors that have contributed to systemic treatment failure are related to mechanisms of chemoresistance such as the presence of unmethylated DNA repair enzyme O6Meg DNA methyltransferase (MGMT) [4].

Although the increased knowledge of molecular alteration in this disease, a lack of success has been reported in different approaches to targeted therapy probably related to the tumoral heterogeneity and signaling-pathway redundancy [5, 6] as well as the absence of a biomarker selection.

All of these considerations should be taken into account in the design of the clinical trials, given that several trials fail to demonstrate a clinical benefit for this disease.

As a result of these difficulties, today the standard of care is a maximal initial resection followed by concurrent radiation and temozolomide.

About 70% of GM will experience recurrence within one year of diagnosis with less than 5% of patients surviving after diagnosis.

In recurrent glioblastoma, there is no standard of treatment. The USA Food and Drug Administration (FDA) has approved bevacizumab (but not by EMA) and TTF.

#### **2. Systemic treatment in newly diagnosed glioblastoma: positive trials**

The EORTC/NCIC clinical trial demonstrated the clinical benefit of adding chemotherapy to the treatment of surgery and radiotherapy in patients with glioblastoma.

In this study, 573 patients were randomized to receive involved-field radiation therapy alone or radiation plus concurrent temozolomide followed up to six cycles of adjuvant temozolomide.

A statistically significant benefit was observed with the addition of temozolomide, with a median overall survival (OS) of 14.6 months vs. 12.1 months [2]. Since the publication of this study, the standard of care (SOC) in newly diagnosed glioblastoma is temozolomide 75 mg/m2 daily during RT followed by 6 adjuvant cycles of 150–200 mg/m2 on days 1-5/28.

*Systemic Treatment in Glioblastoma DOI: http://dx.doi.org/10.5772/intechopen.109243*

A retrospective analysis of 206 patients has been done to determine the MGMT methylation status. In 45% of the cases, MGMT was methylated and the benefit of the treatment with temozolomide was greater (median overall survival 21.7 months vs. 15.3 months).

In non-methylated patients, there was a survival benefit that was not statistically significant [4].

#### **3. Systemic treatment in newly diagnosed glioblastoma: negative results**

Since the publication of the previously mentioned study, by Roger Stupp [2], of what is now the standard of care (SOC), there have been few advances. Furthermore, despite a better understanding of the biology of the tumor, this has not translated into progress in first-line therapy or newly diagnosed GBM. However, this does not mean that efforts to search for new targets and/or therapeutic strategies for improving the prognosis of these patients have been null or void. It must be said that there has been a titanic effort and that the negative results of trials have helped us to steer the research path. Therefore, we are going to review the negative studies with the greatest impact.

#### **3.1 Antiangiogenics**

The rationale for the use of drugs that inhibit vascular endothelial growth factors, such as bevacizumab, was based on the concept that the tumor vasculature could be normalized. This would lead to a decrease in tumor interstitial pressure and, therefore, better access to cytotoxic drugs. Moreover, with increased oxygen supply, the efficacy of radiotherapy would also be improved [7]. On the other hand, it is known that GBM overexpresses vascular endothelial growth factor A (VEGF-A), a key regulator of tumor-associated angiogenesis, and these tumors are highly vascularized [8].

Given that bevacizumab has demonstrated activity in patients with recurrent GBM and there was evidence that indicates the combination of bevacizumab with SOC therapy was active for patients with newly diagnosed GBM, two studies were initiated for first-line patients.

In the **AVAglio trial** [9], 921 patients were randomized to receive bevacizumab (10 mg per kilogram of body weight every 2 weeks) or placebo, plus SOC: radiotherapy (2 Gy 5 days a week; maximum, 60 Gy) and temozolomide (75 mg per square meter of body-surface area per day) for 6 weeks. After a 28-day treatment break, maintenance bevacizumab (10 mg per kilogram intravenously every 2 weeks) or placebo, plus temozolomide (150 to 200 mg per square meter per day for 5 days), was continued for six 4-week cycles, followed by bevacizumab monotherapy (15 mg per kilogram intravenously every 3 weeks) or placebo until progression or unacceptable toxicity. Even though PFS was longer in the bevacizumab group (10.6 months vs. 6.2 months; stratified hazard ratio for progression or death, 0.64; 95% confidence interval [CI], 0.55 to 0.74; P < 0.001), the OS did not differ between groups (stratified hazard ratio for death, 0.88; 95% CI, 0.76 to 1.02; P = 0.10). Maintenance of quality of life and performance status were observed with bevacizumab even though the bevacizumab group had more adverse events (arterial thromboembolic events, hypertension, and complications of wound healing). No predictive influence of MGMT status or any other subgroup variable was observed concerning progression-free survival or overall survival.

The addition of bevacizumab to SOC was also investigated in the Radiation Therapy Oncology Group **(RTOG)-0825 study** [10]. It showed a similar trend toward improvement in PFS (HR 0.79; 95% CI, 0.66 to 0.94; P = 0.007), with a 3.4-month extension of PFS; the difference was not significant according to the prespecified alpha level (P < 0.004) and there was also no statistically significant difference in OS (HR, 1.13; 95% CI, 0.93 to 1.37; P = 0.21).

Finally, the exhaustive review by Cochrane concluded that there is insufficient evidence to support the use of antiangiogenic therapy for people with newly diagnosed glioblastoma [11].

#### **3.2 Integrin inhibition**

Integrins are adhesion molecules involved in several tumorigenic processes such as survival, proliferation, migration, invasion, and angiogenesis [12]. Cilengitide is a selective inhibitor of αvβ3 and αvβ5 integrins that are expressed both in GBM tumor cells and in the vasculature. Moreover, several studies demonstrated a potentiation or synergy when cilengitide is combined with radiation therapy and chemotherapy. That was the rationale for exploring cilengitide in newly diagnosed GBM.

The first trial, **CENTRIC EORTC 26071-22072**, was carried out in methylated MGMT promoter tumors. Cilengitide, 200 mg intravenously twice weekly, was added to SOC, maintenance temozolomide was given for up to six cycles, and cilengitide was given for up to 18 months or until disease progression or unacceptable toxic effects. The primary endpoint was overall survival [13]. Unfortunately, the addition of this drug did not improve the outcome: OS was 26·3 months in both arms (HR,1·02;95%CI,0·81–1·29;p = 0·86) and PFS 10·6 months in the cilengitide arm and 7·9 months in the control arm (HR, 0·92; 95% CI, 0·75–1·12;p = 0·41).

Later, data were published on unmethylated tumors [13]. It offered the opportunity to use dose intensification as a means of overcoming resistance. Patients were randomized to standard cilengitide (2000 mg twice weekly until progression) or intensive cilengitide (2000 mg daily for 5 days during radiotherapy followed by twice weekly until progression) with radiotherapy and temozolomide or a control arm with SOC. Median PFS was 5.6 months and 5.9 months in the standard and intensive cilengitide arms, respectively, versus 4.1 months in the control arm. The median OS was 13.4 months (range, 0–30 mo) in the control arm, 16.3 months (range, 0–29) in the standard cilengitide arm, and 14.5 months (range, 0–29) in the intensive cilengitide arm, which is statistically nonsignificant. No benefit was observed despite dose escalation and most striking was the improvement in OS in patients who were expected to have a worse prognosis as they were unmethylated. The study was underpowered to consider the 3-month improvement in OS was enough. In addition, we do not have a biomarker to select responders.

Integrins are an important target in GBM, but a better understanding of the interaction between the tumor and the extracellular matrix is needed [14].

#### **3.3 PARP inhibitors**

Half of the patients with GBM have methylated MGMT and there is a rationale for combining PARP inhibitors with temozolomide, based on the importance of PARP in mediating basic tissue repair as well as homologous recombination.

The combination of veliparib and SOC did not provide benefits [15]. In ASCO 2022, the **Alliance A071102 trial** was presented [16]. Total of 447 patients with MGMT promoter hypermethylated GBM after radiotherapy and temozolomide were randomized to receive adjuvant temozolomide, given on days 1 to 5 every 28 days, combined with either placebo (n = 224) or veliparib (n = 223), given on days 1 to 7

every 28 days. Treatment was continued for up to six cycles. For phase II, PFS was the primary endpoint. The results were disappointing as the PFS was similar in both groups: 13.2 months with veliparib versus 12.1 months with placebo (HR 1.05, 95% confidence interval 0.86–1.29, p = .31). Median OS was 28.1 months with veliparib and 24.8 months with placebo (hazard ratio 0.89, 95% confidence interval 0.71–1.11, P = .15). The study is negative despite the different hypotheses put forward by the authors about improved survival at intermediate time points.

Effective biomarkers are needed to identify patients who are most likely to benefit from the addition of veliparib.

#### **3.4 ANTI EGFR therapies**

Epidermal growth factor receptor (EGFR) gene amplification on chromosome 7 (EGFR-amp) is expressed in 50% of GBMs. The EGFR variant 3 mutation (EGFRvIII), a tumor-specific deletion of exons (2–7), is active and is observed in approximately 50% of GBMs with EGFR (~25% overall) [17]. Nevertheless, EGFRtargeted treatments in GBM have been disappointing.

#### **3.5 Antibody-drug conjugate**

Depatuxizumab mafodotin (depatux-m) is an antibody-drug conjugate composed of a monoclonal antibody that binds to activated EGFR and is bound to a microtubule inhibitor toxin. It was tested in a phase III trial, adults with centrally confirmed, EGFR-amp newly diagnosed GBM [18]. Patients were randomized to receive SOC plus depatux-m at 2.0 mg/kg during RT, then 1.25 mg/kg on days 1 and 15/28, and continue until disease progression versus SOC. The trial was a phase III with OS as the primary endpoint. There was no improvement with the addition of the antibody; OS for depatux-m over placebo (median 18.9 vs. 18.7 months, HR 1.02,95% CI 0.82–1.26, 1-sided p = 0.63). PFS was longer for depatux-m than placebo (median 8.0 vs. 6.3 months; HR 0.84, 95% confidence interval [CI] 0.70–1.01, p = 0.029), particularly among those with EGFRvIII-mutant (median 8.3 vs. 5.9 months, HR 0.72, 95% CI 0.56–0.93, 1-sided p = 0.002) or MGMT unmethylated (HR 0.77, 95% CI 0.61–0.97; 1-side p = 0.012) tumors but without an OS improvement. One of the most peculiar toxicities of this drug is the corneal epitheliopathy that occurred in 94% of depatuxm-treated patients (61% grade 3–4), causing 12% to discontinue.

#### **4. Pharmacologic treatment of recurrent glioblastoma**

In the recurrence set, the prognosis of these patients is poor, with an estimated survival of about 6 months [19].

Compared to newly diagnosed glioblastoma, the management of the recurrent disease is not curative and less standardized without randomized trials. Different approaches should be considered including systemic agents (chemotherapy and target therapy) or locoregional treatments (radiation therapy and surgery) [20].

There is limited evidence for the systemic therapy of recurrent GB (rGB).

Several prognostic factors should be taken into account to select the patients that can benefit from systemic treatment after recurrence. Some of these factors are tumor size, location, performance status, and administration of steroids.

It is recommended to enroll these patients in a clinical trial whenever possible.

Outside a clinical trial, a second-line treatment could be considered.

The most commonly used agents are nitrosoureas, bevacizumab, and temozolomide, but none is approved by EMA because most of the time the evidence was derived from small to no randomized studies [21]. Bevacizumab has been approved by the FDA for recurrent high-grade glioma.

Treatments as promising as immunotherapy and drugs against EGFR are not superior to the treatments cited. Several novel treatments are undergoing evaluation in clinical trials.

#### **4.1 Nitrosoureas**

Nitrosoureas (lomustine, carmustine, fotemustine) have shown activity in phase II trials in rGB.

Lomustine (CCNU) is an oral nitrosourea. It has shown a modest improvement in overall survival (median OS 7.1–9.8 months).

Lomustine has never been shown to be superior to other drugs in randomized studies but represents the control treatment arm in randomized clinical trials. Lots of new drugs, especially multitarget tyrosine kinase inhibitors, have been tried alone or in association with lomustine or against lomustine without any benefit in overall survival [22].

Fotemustine is a new intravenous nitrosourea with a better toxicity profile. It has proven activity in glioblastoma in several phase II studies and is mainly used in Europe [23].

#### **4.2 Antiangiogenic agents**

Bevacizumab (BV) is a VEGF-A (vascular endothelial growth factor A) targeting monoclonal antibody. It was a very promising agent in rGB. In phase II, studies showed a PFS-6 rate ranging from 18–42% and median OS from 6.5 to 9.2 months. However, in randomized clinical trials, it has not been proven to have better results than lomustine.

In the phase II randomized trial BELOP (5) the combination of BV and lomustine showed an OS benefit over lomustine. Nevertheless, the phase III EORTC 26102 study randomized more than 400 patients with rGB to BV plus lomustine versus lomustine. The results showed a significant difference in PFS, but without any impact on OS, which was the main endpoint [24]. It is not yet known, which subgroup of patients could benefit from BV and its real impact on OS. Combinations of BV with other agents do not appear to be superior to monotherapy.

Regorafenib, an oral multi-kinase inhibitor, has been investigated versus lomustine in the randomized phase II trial REGOMA. The primary endpoint was overall survival in the intention-to-treat population, which was higher in the experimental arm. However,the planned statistical design did not have enough power to estimate survival advantage. Therefore, the authors concluded that phase III is needed to confirm this benefit [20].

#### **4.3 Temozolomide**

Temozolomide rechallenge can be considered an option in patients who have tumor recurrence beyond four to six months from the end of the first-line treatment with temozolomide and have a methylated MGMT promotor.

Another strategy consists of the administration of temozolomide in an extended regimen. Extended schedules had been developed to overcome TMZ resistance in phase II studies [25].

There are small studies that yield modest results in rGB (PFS-6 rates 17–50%) [21].

#### **4.4 AntiEGFR therapy**

About 50% of all GB patients present an amplification of the epidermal growth factor receptor (EGFR) gene. Agents targeting this receptor failed to show a significant survival impact on patients with rGB.

The most promising agent has been depatuxizumab mafodotin, an antibody-drug conjugate, that consists of an antibody directed against EGFR and EGFRvIII, conjugated to a toxin (monomethyl auristatin F). The INTELLANCE-2 /EORTC 1410 phase II randomized study [26] investigated depatux-M in combination with temozolomide or as a single agent in recurrent EGFR amplified GB. Patients who received depatux-M and temozolomide had a trend toward improved survival but did not reach statistical significance.

#### **4.5 Future promising agents**

It is necessary to improve the design of clinical trials in GB.

Personalized treatments based on the tumor's molecular characteristics have had promising results.

There are small studies with inhibitors of NTRK (neurotrophic tropomyosin receptor kinase), BRAF (B-Raf proto-oncogene), FGFR (fibroblast growth factor receptor), PDGFR (platelet-delivered growth factor receptor), IDH (isocitrate dehydrogenase), and histones, mainly, that are showing interesting preliminary results.

Other types of immunotherapy, such as chimeric antigen receptor T-cells (CAR-Ts), chimeric antigen receptor macrophages (CAR-Ms), oncolytic viruses, and vaccines, are under evaluation [27].

#### **5. Recurrence glioblastoma: radiotherapy and surgery**

Despite systemic treatment, other options could be considered for recurrence.

As previously referred, in this context, treatment decisions must be individualized.

One of the most important prognostic factors for benefit from local treatment is the previous performance status. Other useful factors include young age, the extent of the disease, the histologic grade, the relapse-free interval, the recurrence pattern (i.e., local versus diffuse), and the extent of the second surgical resection [28].

A negative factor is ependymal involvement, which is independent of performance status, tumor size, and extent of resection [29].

Patients with a localized recurrence are better candidates for reoperation or reirradiation interventions than those with primary refractory disease or diffuse or multifocal relapse.

It is important to point out that these patients should be referred to a multidisciplinary brain tumor center with a multidisciplinary team to revise images, evolution, and options of treatment [28, 30, 31].

In conclusion, the best candidates for reoperation are patients with large but wellcircumscribed, symptomatic tumors that are amenable to complete or near-complete resection, particularly if the tumor has recurred after an extended interval.

The benefit of reirradiation of glioblastoma is uncertain and can be considered in selected patients. Occasionally used in patients with a localized or out-of-field glioblastoma recurrence. Instead, patients with a poor performance status have poor prognostic, and the risks of receiving subsequent treatment outweigh the benefits [32, 33].

#### **5.1 Reoperation**

Approximately, only 20 to 30 percent of patients with recurrent glioblastoma are candidates for a re-operation [19, 34, 35].

The technics used are the same as for primary resection and included 5-aminolevulinic acid (5-ALA) guided resection that showed benefit in recurrent glioblastoma [36–38].

There is no evidence to suggest that these results are better than and can be expected with radiation and/or chemotherapy alone.

Two meta-analyses have analyzed surgery as a treatment approach in recurrent glioblastoma.

The first study assessed eight observational studies for a total of 1906 patients with glioblastoma who underwent primary surgery and 709 patients with secondary surgery. The pooled hazard ratio (HR) showed a longer OS for patients receiving surgery at the time of recurrence (HR: 0.722; p: 0.001).

The second meta-analysis selected nine studies for a total of 1507 patients with glioblastoma and 1335 patients treated with re-intervention.

Among these studies, OS after repeat surgery ranged from 8 to 13 months. Maximal safe resection appears to confer a significant OS benefit (HR 0.59, p: 0.1). Radiographic confirmed gross total resection was the most prognostic variable related to the extent of surgery and was associated with longer OS (HR 0.52, p: 0.01) [39].

Another interesting option is carmustine polymer wafers. A review revealed three trials in which patients with glioblastoma who received carmustine wafers had statistically significant longer overall survival. Overall results of these trials seem to suggest that carmustine wafer implantation demonstrates promise as an effective and tolerable treatment strategy for GBM [40]. Daily practice is not commonly used due to potential surgical complications.

In conclusion, surgery should be included in the treatment algorithm for recurrent glioblastoma. It should be proposed when it is technically safe and associated with a feasible total resection, especially in patients with good performance status. Optimal management after surgery is still unknown, and prospective studies are ongoing to study different strategies, such as RESURGE trial [41].

#### **5.2 Reirradiation**

Salvage reirradiation has been utilized in the treatment of recurrent diseases for years. The role of reirradiation in patients with recurrent glioblastoma is uncertain, and there is little prospective data. For this reason, participation in clinical trials is encouraged.

As most recurrences occur within the high-dose radiation field (90–95%), reirradiation is generally poorly considered as a treatment option due to the high risk of toxicity.

#### *Systemic Treatment in Glioblastoma DOI: http://dx.doi.org/10.5772/intechopen.109243*

The adequate selection of patients suitable for reirradiation is a key issue. Age, performance status, target volume, time to progression, type of progression, and site of recurrence are essential elements to consider. Different techniques can be used: conventionally fractionated radiotherapy (RT), hypofractionated stereotactic radiosurgery (HFSRT), and stereotactic radiosurgery (SRS) [42].

Based on mostly retrospective series, selected patients with small recurrent tumors and a good performance status may benefit from repeat radiation using modern highprecision techniques to deliver total doses of 30 to 35 Gy in 5 to 15 fractions [43].

Reirradiation with conventional involved field radiation at therapeutic doses (54 to 60 Gy) is not recommended in patients with relapsed disease due to treatmentrelated toxicity. The most common form used is fractionated radiosurgery or hypofractionated radiotherapy (e.g., 30 to 35 Gy in 5 to 15 fractions). Selection is based on the preference of the treating radiation oncologist and local availability since there are no clear differences in efficacy [44].

Reirradiation can be given with both concurrent or sequential administration of systemic therapy (TMZ, bevacizumab, and immunotherapy). The available data in patients with recurrent glioblastoma generally suggest that reirradiation modestly improves progression-free survival compared with systemic therapy alone, but overall survival is similar [45].

A few prospective data are available in a phase II trial 182 patients with recurrent glioblastoma were randomized to receive bevacizumab alone or in combination with radiation treatment (35 Gy in 10 fractions). The combination of radiation therapy and bevacizumab prolonged the PFS of these patients without significant improvement in OS [45].

The risk of radionecrosis should also be considered [46].

The benefit of the addition of bevacizumab to radiotherapy treatment was published in a recent systematic review. Data from a total of 1399 patients, were analyzed (954 patients receiving RT alone and 445 patients receiving RT and bevacizumab). Multivariate analysis showed that bevacizumab was associated with significantly improved. Patients receiving BVZ also had significantly lower rates of radionecrosis (2.2% vs. 6.5%) [47].

Other initial trials (phase I) studied the combination of RT, bevacizumab, and immunotherapy with promising results, but further controlled studies are needed to confirm these effects [48, 49].

Interstitial brachytherapy has been used in patients with recurrent high-grade gliomas, with several observational studies suggesting a survival benefit. However, brachytherapy is associated with a high incidence of radiation necrosis [50, 51].

An alternate form of brachytherapy uses an inflatable balloon catheter containing a liquid I-125 radioisotope (GliaSite) inserted at the time of surgical resection, which allows delivery of a quantifiable high dose of radiation to the tissue. No randomized clinical trials have been reported comparing this form of brachytherapy with other approaches. The role of brachytherapy is diminishing as experience with SRS and fractionated localized limited field radiation evolves [52, 53].

#### **6. Immnunotherapy**

Historically, the central nervous system (CNS) was considered to be immunologically isolated. However, today we know that the immunity system of the CNS is different but not incapable. There are functional lymphatic vessels and there are antigen-presenting cells: microglia, macrophages, astrocytes, and classical APCs such as dendritic cells [54].

Glioblastoma is a cold tumor, with a low mutational burden; furthermore, as detailed below, it has demonstrated a poor response to immune stimulation therapies, such as immune checkpoint blockade. Even when T-cell responses are induced in CNS tumors by means such as vaccination, as discussed above, the number of antigen-specific TILs can remain relatively low, and the cells that are present often show a depleted phenotype. The reduced number and limited activity of T-cells in CNS tumors are largely due to the unique immunosuppressive immune environment of the brain [55].

The final step of the immune response in glioblastoma is the destruction by the active T-lymphocyte of the GBM cells after binding to their tumor antigen on MHC-I *via* the T-cell receptor (TCR). These T-cells are activated after recognizing the GBM cells, secreting inflammatory cytokines, and inducing GBM cell death.

Glioblastomas are characterized as tumor with a low median TMB and a lack of infiltrating lymphocytes [56]. Current approaches focus on: (1) Increasing glioma immunogenicity and activating the adaptative immune response by using tumor vaccines and oncolytic viruses, (2) Revert T-cell energy and promote a more inflamed tumor microenvironment by using immune cytokines, chemokines, and cytokine modulators, and (3) Overcome the lack of resident tumor infiltrating lymphocytes by directly engaging T-cells through direct activators such as CAR T-cells, TCBs, and bispecific T-cell engager antibodies.

#### **6.1 Checkpoint inhibitor**

GBM overexpresses PDL1, leading to PD-L1 binding to PD-1 and thus inhibiting the immune response [55].

Treatment with immune checkpoint blockade has shown improved survival in murine glioma models. However, data from phase III studies with the anti-PD-L1 nivolumab did not meet their primary endpoint of OS in the final analysis.

For newly diagnosed patients with MGMT-methylated or indeterminate GBM, the SOC therapy was compared with the same scheme plus nivolumab [57]. The trial included 716 patients who were required to have a centrally assessed methylated MGMT promoter, a Karnofsky performance status (KPS) of ≥70, and ≤ 3 mg dexamethasone at baseline. This study had two primary endpoints: PFS and OS. Regrettably, there were no significant differences observed for the 2 primary endpoints of the study. The median PFS for patients on the nivolumab arm was 10.6 months, compared with 10.3 months for the control arm. For patients not on corticosteroids, the median OS was 31.3 months for the nivolumab arm and 33.3 months for the control arm.

Nivolumab was also investigated for MGMT unmethylated GBM [58]. The trial compared nivolumab concurrent with RT followed by nivolumab until disease progression or unacceptable toxicity versus SOC. The addition of nivolumab did not improve efficacy. A total of 560 patients were randomized; median OS was 13.4 months (95% CI, 12.6–14.3) with NIVO+RT and 14.9 months (95% CI, 13.3–16.1) with TMZ + RT (HR, 1.31; 95% CI, 1.09–1.58; P = 0.0037). Median PFS was 6.0 months (95% CI, 5.7–6.2) with NIVO+RT and 6.2 months (95% CI, 5.9–6.7) with TMZ + RT (HR, 1.38; 95% CI, 1.15–1.65). A subgroup analysis based on established prognostic factors, including age, KPS, and degree of surgery, showed no significant benefit for the addition of nivolumab in any patient subgroup. One interesting feature was the baseline PD-L1 expression in tumor tissue: <1% in >55% of RT-TMZ and > 62% of RT-nivolumab patients. Although debate still rages regarding the role

#### *Systemic Treatment in Glioblastoma DOI: http://dx.doi.org/10.5772/intechopen.109243*

and predictive value of this biomarker as well as optimal threshold, such a high level of lack-of expression of a key mechanistic molecule is worrisome.

Limitations of immune-based therapy may be related to tumor-associated factors, such as poor immunogenicity and tumor-induced immune tolerance, but it is important that treatment (SOC) induced immune regulatory effects may also play major roles, both adversely and beneficially [54]. In recurrent glioblastoma:

The CheckMate 143 trial included Cohort 1 in which patients with refractory glioblastoma were randomized to nivolumab monotherapy at 3 mg/kg every 2 weeks (10 pts), or nivolumab 1 mg/kg + ipilimumab 3 mg/kg every 3 weeks (10pts) and a non-randomized cohort 1b of 20 pts. that received nivolumab 3 mg/kg + ipilimumab 1 mg/kg Q3W for 4 doses, followed by nivolumab 3 mg/kg Q2W. A total of 3 pts. achieved a partial response (1 pt. with monotherapy and 2 with the combination). Based on the similar OS, RR and the lower degree of G3/4 toxicity, monotherapy with nivolumab was chosen as the treatment strategy for further development.

In the phase III trial, 369 patients were randomized to receive either nivolumab 3 mg/ kg Q2W or bevacizumab 10 mg/kg Q2W. This study was negative for its principal objective of overall survival (OS of 9.8 months vs. 10 months and hazard ratio 1.04). OR and PFS favored bevacizumab, but ORR in the nivolumab was 7.8% and the duration of response was better than the one achieved with bevacizumab (11.1 versus 5.3 months) [59].

Other similar trials with checkpoint inhibitor monotherapy worth mentioning are the KEYNOTE-028 trial and the NCT01375842 trial of Atezolizumab. The KEYNOTE-028 [60] included 26 pts with bevacizumab-naïve recurrent glioblastoma have received pembrolizumab 10 mg/kg Q2W for a maximum of 24 months. 2 patients achieved partial responses (ORR of 7.6%) that lasted 8.3 and 22.8 months respectively and the 6 months PFS was 37.7%. On the NCT01375842 [61], 1 of 16 patients with recurrent glioblastoma showed a partial response (ORR of 6.25%).

The fact that in the Checkmate 143 trial nivolumab achieved similar outcomes to Bevacizumab (which is considered an active treatment in the recurrent setting), and the preclinical found that VEGF can mediate immunosuppression on the tumor stroma, the combination of immunotherapy with antiangiogenics could be worth investigating in the recurrent GBM setting. Three different clinical trials that have combined pembrolizumab with bevacizumab [62], avelumab with axitinib [63] or durvalumab with bevacizumab [64], have failed to show better results to those reported with antiangiogenic monotherapy.

Only a selected number of patients with recurrent glioblastoma will be considered candidates for a secondary resection, and as a consequence, the studies in the neoadjuvant setting have included a small number of patients. One small study with nivolumab [65] showed an increase in infiltrating T cells and an increase in the IFN response, while a similar one with pembrolizumab [66] failed to show any changes in the number of CD8 positive cells.

Intriguingly, the duration of response in some patients was high, but the expression of PD1 or the presence of hypermutation has not been associated with response to immunotherapy in gliomas.

#### **6.2 Oncolytic viruses and tumor vaccines**

Apart from the direct tumor cell killing that occurs after tumor cells are infected, oncolytic viruses have the potential of increasing immunogenicity in glioblastoma by delivering PAMPs (pathogenic associated molecular patterns) and facilitating the release of tumor-antigens by dying virus-infect cells.

#### *Glioblastoma - Current Evidence*

HSV is the most studied virus in immunotherapy [67]. It can function as both an oncolytic agent and a transgene vector, which can be armed with immunomodulatory or angiogenic modulatory genes (i.e., GM-CSF in TVEC and IL2 in G47delta).

Results with G47delta injected intratumorally in patients with recurrent glioblastoma have shown issues with immediate enlargement of the contrast-enhanced area of the target lesion on MRI caused by the treatment, that is produced tumor destruction and lymphocyte infiltration, but the results in terms of OS (1-year OS of 38.5%) and especially the presence of a subset of patients with longer OS seem promising [68].

To create the DNX-2401 adenovirus, a 24-base pair deletion in the E1A gene that renders the virus unable to infect non-tumoral cells was introduced alongside an RGD-motif that enables the virus to infect integrin-rich cells, that are enriched in the tumor cells. Preliminary results of a phase I study show that 20% of patients with recurrent glioblastoma treated with intratumoral injection achieve OS 3y [69].

Another pilot study in patients with pediatric DIPG (a terrible disease where the historic series show a median OS of around 12 months) showed a reduction in tumor size in 9/12 patients, a 25% OR, and a median OS of 17.8 months [70].

Poliovirus PSRIVO is introduced in the tumor area through convention-enhanced delivery and recognizes the poliovirus receptor CD155, which is widely expressed in neoplastic cells in comparison with normal tissues. In a dose-finding study that included 61 patients with glioblastoma, also benefited a subset of patients (21%) that achieved long-term control at 24 and 36 months [71].

Compared with other tumors, the low TMB burden leads to a lower potential of Tumor-specific antigens (TSAs), mutant proteins expressed exclusively in tumor cells. Most studies using peptidic vaccines have focused either on personalized vaccines, with only two small pilot projects being published [72, 73], or vaccination against Tumor-associated antigens(TAAs), proteins present in normal tissues but overexpressed in tumors. A vaccine, called rindopepimut, designed to target the EGFRvIII, which is present in 30% of GBM cases was recently tested in a randomized phase III, after showing immunogenicity, safety, and activity in earlier clinical trials [74]. A phase III study, that randomized 745 patients that had completed their initial chemoradiation without progression showed negative results for OS (20.0 vs 20.1 m) in both patients with and without residual disease [75].

In comparison with peptidic vaccines, DC vaccines have the potential of being generated directly from coculture with tumor lysates, allowing the co-targeting of both TSAs and TAAs. Preclinical studies on glioblastoma have demonstrated that DC vaccines can reduce tumor growth, prolong survival and induce tumor-specific IFN-γ, and cytotoxic T-lymphocytes responses associated with T-cell infiltration of tumors.

Several small clinical trials have shown that this approach is safe and feasible [76–78], and the preliminary results of the largest clinical trial to date, testing DC-Vax, vs placebo with crossover at progression in 331 patients seem promising. But the final unblinded results have yet to be published [79]. Another potential source of dendritic vaccines is the ones generated by exposing cells to pp65, which is a major structural protein of CMV a virus that is frequently present in glioblastoma cells. Although studies using pp65 vaccines are small the median PFS of 25.3 m and OS of 41.1 m are intriguing [80].

#### **6.3 Immunocytokines, chemokines, and other cytokine modulators**

Immunocytokines are molecules that target immunostimulating cytokines such as TNF or IL-2 to the tumor microenvironment using signals that direct them to the tumor cells, immune infiltrating cells, or components of the tumor stroma. One

#### *Systemic Treatment in Glioblastoma DOI: http://dx.doi.org/10.5772/intechopen.109243*

potential therapeutic agent in this class is L19TNF, a multimer of TNF fused to the antibody L19 that binds a tumor-specific epitope of the extracellular matrix protein fibronectin. Preliminary studies have shown the safety, feasibility, and intriguing preliminary clinical results in both combinations with lomustine in refractory GBM, and combination with chemoradiation in front-line patients.

TGF-B upregulation in glioblastoma has been linked to increases in the migratory potential, promoting EMT, inducing a CSC-like drug-resistant phenotype, and an immunosuppressive microenvironment [81]. Despite some patients treated with TGFB inhibitors in clinical trials showing prolonged responses [82, 83], the overall results with oral TGFBR have been disappointing [84] most likely due to insufficient target inhibition due to concerns over cardiotoxicity.

CSF-1R inhibitors are cytokine modulators that try to repress tumor-associated myeloid cells that form a substantial proportion of the immunosuppressive glioblastoma microenvironment, by downregulating CSF1R, an important receptor for macrophage differentiation and survival. However clinical trials both in first-line patients combined with chemoradiation and in patients with recurrent disease in both monotherapy and combination with checkpoint inhibitors show limited clinical efficacy.

#### **6.4 CAR T-cells, TCBs, and bispecific T-cell engager antibodies**

CAR-T cell treatment share with vaccines the necessity of identifying targets that are primarily present in tumor cells with low expression in normal tissues (TAAs/TSAs).

Accordingly, EGFRvIII has also been chosen as a target for CAR T-cell treatment. One potential issue is that although most patients treated with CAR T-cells developed noticeable peripheral levels of EGFRvIII-directed CAR T-cells when their tumors were resected half of the patients had lost their baseline expression of EGFRvIII [85].

Subsequent small trials with second and third-generation trials including expression of costimulatory proteins show only minimal signs of activity in a few patients [86].

IL13Rα2 CAR-T has also been tested in small clinical trials, with some patients achieving clinical benefit, including 1 patient presenting a complete response. Finally.

HER2 CAR-T cells were deemed to be safe in the phase I clinical trial that included 17 patients, including 1 patient with partial response and 3 with disease stabilization for more than 4 months [87].

Another potential way to overcome the lack of antigen presentation and infiltrating T-Cell is by the use of bispecific antibodies that target at the same time a target present in immune cells, that many times is CD3 and a TSA or TAA. Several modifications to the bispecific antibody structure can be made to modify its protein and characteristics. For example, AMG 596 is composed of two single-chain variable fragments one binding to CD3, and the other to EGFRvIII, while RO7428731 contains both variable regions against EGFRvIII and CD3 and an IgG structure [88].

*Glioblastoma - Current Evidence*

#### **Author details**

María Ángeles Vaz1 \*, Sonia Del Barco Berrón2 , Raquel Luque3 , María Villamayor1 , Juan Manuel Sepúlveda Sánchez4 and María Vieito5

1 Ramón y Cajal Universitary Hospital, Madrid, Spain

2 Josep Trueta Universitary Hospital, Gerona, Spain

3 Virgen de Las Nieves Universitary Hospital, Granada, Spain

4 12 Octubre Universitary Hospital, Madrid, Spain

5 Vall D'Hebron Universitary Hospital, Barcelona, Spain

\*Address all correspondence to: mavaz4@gmail.com

© 2022 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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#### **Chapter 6**

## Noncanonical (Non-R132H) IDH-Mutated Gliomas

*Tariq D. Al-Saadi and Roberto J. Diaz*

#### **Abstract**

Mutations in *IDH1* or *IDH2* confer a significant survival advantage compared to their isocitrate dehydrogenase (IDH) wild-type counterparts and, as such, are the most significant prognostic factors in this group. The mutations in the IDH1 gene are heterozygous and almost always involve only a single residue (arginine 132), which is replaced by histidine in roughly 90% of tumors. Regardless, the non-p.R132H (noncanonical) mutations in the IDH1 gene were also documented in around 20% of mutated glioma. The noncanonical IDH mutations have distinguishing radiological and histological features. The existence of such tumors seems to be associated with a genetic predisposition to cancer development.

**Keywords:** noncanonical, IDH-mutant, glioma, astrocytoma

#### **1. Introduction**

#### **1.1 The 2020 WHO classification of adult gliomas**

According to the latest World Health Organization (WHO) classification of CNS tumors, all isocitrate dehydrogenase (IDH)-mutant tumors were classified as either IDH-mutant oligodendrogliomas or astrocytomas and graded as WHO grade 2, 3, or 4 [1]. This classification recognized a grade 4 IDH-mutant astrocytoma and tumors harboring CDKN2A/B homozygous deletion. We favor using the term cancerous glioma to the previous term "Low-Grade Glioma (LGG)," since the current 2020 WHO classification presented major changes that advance the role of molecular diagnostics in CNS tumor classification. With the new classification, there are three groups of adult-type gliomas (**Figure 1**).

Group 1 is the astrocytoma with IDH mutation, group 2 is astrocytoma with no IDH mutation (IDH wild-type), and group 3 is oligodendroglioma, which carries the IDH mutation and 1p/19q co-deletion. Other significant molecular profile findings include IDH1, IDH2, ATRX, TP53, and CDKN2A/B. Group 1 is further classified based on the histopathological grade into 2, 3, and 4. The second group is the astrocytoma with IDH wild-type status, where only one histological grade is given (grade 4) due to the nature of the disease and it is prognosis. A third group is oligodendrogliomas which are characterized by the 1p/19q co-deletion which is unique for this group and considered to be a positive prognostic marker for this particular group [2, 3].

**Figure 1.**

#### **2. IDH mutation in glioma**

IDH1 mutations are very recurring in World Health Organization (WHO) grade II astrocytomas, anaplastic astrocytomas WHO grade III, low-grade/anaplastic oligodendrogliomas, and secondary glioblastomas (representing 80%, 64%, 66–80%, and 83%, respectively) [4–7]. Several studies have consistently reported a positive association between the IDH1 mutations and the better prognosis for patients with malignant gliomas [7–11]. Yet, for the adult cancerous glioma or (LGGs) vague results have been published so far. Metellus et al. studied a small series of 47 LGGs (85% oligodendroglial and 15% astrocytic tumors) and deduced that IDH1 mutations were positive prognostic values and associated with more prolonged overall survival (OS) and progression-free survival (PFS) [12]. Dubbink et al. analyzed a retrospective series of 49 low-grade astrocytomas for IDH1/2 mutations and found a highly significant correlation between OS and IDH1 status [13]. A more considerable series by Sanson and his colleagues with 100 LGGs (88% oligodendroglial tumors and 12% astrocytomas WHO-II) concluded that IDH1 is a prognostic marker and associated with longer OS only but not with PFS [9]. An association between IDH1 mutation status and OS was also noted in a cohort of 139 LGGs consisting of 61 oligodendroglial and 78 astrocytic tumors [5]. Contrariwise, additional investigations on patients with LGGs did not report any correlation between IDH1 mutation and OS [14–16]. These studies were associated with low hazard ratio (HR); however, a meta-analysis reported later (included the later studies) showed a positive HR between the IDH mutation status and death (with less mortality rate in IDH-mutated glioma compared to the wild-type) [17].

#### **2.1 Biological impact of IDH mutation**

In adults, mutations in *IDH1* or *IDH2* confer a significant survival benefit compared to their IDH wild-type counterparts and as such are the most important prognostic factor in this group [9]. Glioma-specific mutations in IDH1 always affect the amino acid arginine in position 132 of the amino acid sequence which belongs to

#### **Figure 2.** *Biological role of IDH1 mutation [22].*

an evolutionary highly conserved region located at the binding site for isocitrate [8]. The role of IDH1 mutations in tumor biology currently is intensely studied. Mutations inactivate enzyme activity and confer the novel function of catalyzing the conversion of alpha-ketoglutarate (αKG) to D-2-hydroxyglutarate (2HG) [5]. The downstream effects of mutant IDH include decreased cellular NADPH and αKG levels, HIF1a stabilization, increased production of 2HG, which competitively inhibits αKG-dependent enzymes such as histone methyltransferases and 5-methylcytosine hydroxylases [18–22], as shown in (**Figure 2**).

### **3. Noncanonical IDH-mutant gliomas**

### **3.1 Overview and prevalence**

Mutations in the IDH1 gene are heterozygous and almost always affect only a single residue (arginine 132), which is replaced by histidine in roughly 90% of tumors [4, 9, 23–25]. Nonetheless, non-p.R132H mutations in the IDH1 gene (e.g. p.R132C) have been documented to accumulate at higher frequencies in histological subtypes of glioma [5] in astrocytomas of Li-Fraumeni patients [26] and in patients with AML [27]. Visani and his co-authors found that around 19% of grade II or III tumors harbored a noncanonical IDH mutation, while in GBM they recognized only the IDH1-R132H mutation [28].

Blass et al. [24] sequenced 685 primary brain tumors to analyze the genomic region spanning wild-type R132 of IDH1. They recognized 221 somatic IDH1 mutations with higher frequency in secondary glioblastomas followed by oligoastrocytomas,

oligodendrogliomas, and diffuse astrocytomas (88%, 78%, 69%, and 68% respectively). Exclusively one wild-type allele was detected, and all the mutations were heterozygous. Mutation in codon 132 of IDH1 was detected only and 205 mutations were of the R132H type. Nevertheless, they also encountered leading to R132C, R132S, R132G, R132L, and R132V (eight, four, two, one, and one mutation, respectively), as shown in **Figure 3**. There was no apparent association of the rare mutation types with a distinct tumor entity, although six of the eight R132C mutations were seen in astrocytomas.

Hartmann and his colleagues analyzed 1010 human gliomas for mutations in codons 132 and 172 in the genes for IDH1 and IDH2, respectively [5]. Their series consisted of 1010 diffuse gliomas including diffuse astrocytomas WHO grade II (227), anaplastic astrocytomas WHO grade III (228), anaplastic oligoastrocytomas (177), anaplastic oligodendrogliomas WHO grade III (174), oligodendrogliomas WHO grade II (128), and oligoastrocytomas WHO grade II (76). R132H was the dominant amino acid sequence alteration accounting for 92.7% of the detected mutations followed by R132C, R132S, R132G, and R132L. The type and distribution of the mutations are given in **Figure 4**.

The disparities in the literature regarding the low frequencies of R132S, R132G, and R132L may be due to dissimilarities in sample size and different types of tumors examined. Franceschi and his colleagues lately reviewed 390 patients with an R132H-IDH1 mutation and 34 patients with a non-R132H mutation [29]. Likened to patients with the R132H-IDH1 mutation, patients with non-R132H mutations were discovered to have less frequent 1p19q co-deletion. In addition, they were also younger than those with noncanonical IDH1 mutation (p < 0.001). Improved overall survival was correlated to the extent of surgical resection, 1p19 co-deletion existence, and the presence of non-R132H mutation [29].

The prognostic impact of non-R132 mutation is still under study and not fully defined.

**Figure 4.**

*Type of 716 IDH1 and 31 IDH2 mutations and frequency among mutations in 1010 WHO grades II and III astrocytomas, oligodendrogliomas, and oligoastrocytomas analyzed by Hartmann et al. [5].*

Since most mutations affect the same hot spot region of IDH1-R132H, it was naturally presumed that the associated clinical outcomes are similar to that of IDH1- R132H mutated tumors, as implied by the difficulty in culling clinical data for specific noncanonical mutations from documented series. Restricted data are available in the literature concerning the prognosis, overall survival, and role of adjuvant therapies (radiotherapy and/or chemotherapy) in patients with noncanonical IDH mutations. Moreover, the literature also lacks studies that distinguish the IDH-mutant astrocytomas and oligodendrogliomas. The issue with combining the two different groups is that the oligodendrogliomas are characterized by the 1p19q co-deletion with the positive prognostic marker for this group. This might have an influence on the overall conclusion of these studies.

**Figure 5a-d** displaying a Scopus review of documents that cited the "noncanonical IDH" OR "non-R132" mutated glioma in the title or abstract. There were 10 total articles. The figure shows the overlay visualization of the authors and the connections between the authors. The colors demonstrate the year of publication, and the size of the circle displays the weight of the author in terms of the number of published documents in this domain. The lines indicate the connectivity between the authors. For instance, Franceschi [29] was involved in 3 documents and had a total of 17 connections (**Figure 5b**). The same applies to Brandes (**Figure 5c**) [30]. Nevertheless, Angelini [31] for instance was involved in one document only (smaller circle) and had only a total link of eight (**Figure 5d**).

#### **3.2 Age distribution in noncanonical IDH-mutated glioma**

There were only three articles that reported the significance of the age distribution in the IDH-mutated gliomas [5, 29, 32]. Posetsch and Franceschi and their colleagues reported a younger age for patients harboring all types of IDH1 noncanonical mutations as compared to IDH1 canonical mutation (median age 35 vs. 43 years and

**Figure 5.** *The overlay visualization of noncanonical IDH-mutated article's authors (Scopus indexed).*

29 vs. 39 years) [29, 32]. Yet, Hartmann et al. reported a significantly younger age only for patients with IDH1 R132C noncanonical mutations with a median age of 34.9 vs. 42.9 years [5].

#### **3.3 Patient outcome**

A recently published systematic review and meta-analysis aimed to evaluate the clinical role of IDH noncanonical mutations documented a possible favorable prognostic role for IDH noncanonical mutations [33]. Another study reported a prolonged survival for patients with IDH1 noncanonical mutations as compared to IDH canonical mutation [29]. However, two other studies reported no association between the noncanonical mutations and the survival rate [23, 32]. Nevertheless, the later studies were lacking the reporting of the survival hazard ratio (HR) with the confidence interval.

#### **3.4 Current therapy and future direction**

One of the most remarkable phenomena noticed in IDH-mutated glioma is the production of 2HG. This oncometabolite was found to be involved in the activation of different cancer-associated signaling pathways in addition to tumorigenesis and tumor progression.

Targeting the mutant enzymes of the IDH1/2 has long been sought as a novel therapeutic strategy to prevent the progression of cancers harboring the IDH1/2 mutation [34]. The benefit of this targeted therapy in glioma using small-molecule inhibitors have been established by several continuing investigations [35, 36]. An example of IDH-R132H enzyme inhibitor is the compound AGI-5198, which is an allosteric, selective inhibitor inhibiting the synthesis of 2HG in mouse and human glioma cells [36, 37]. Researchers also found that mutant IDH1 promotes selective vulnerability by altering NAD+ supply [38]. The expression of Naprt1 (a rate-limiting enzyme within the NAD+ salvage system) can be reduced by the introduction of

*Noncanonical (Non-R132H) IDH-Mutated Gliomas DOI: http://dx.doi.org/10.5772/intechopen.105469*

mutant IDH1 and results in more depressed basal NAD+ levels. Exposure to NAMPT inhibitors thus effectively hinders both NAD+ salvage pathways in IDH1-mutant cells, resulting in a metabolic crisis that activates the energy sensor AMPK and initiation of autophagy. They also highlighted that reduced NAD+ salvage plays a major role in the mechanism of NAMPT inhibitor hypersensitivity [38].

Ongoing phase I/II clinical trials are currently in progress to assess the safety of different IDH-mutant inhibitors in glioma patients. Early clinical results suggest that the IDH1-mutant inhibitor AG-120 (ivosidenib) is an example of an IDH1-mutant inhibitor that is satisfactorily accepted in patients with previously treated noncontrast-enhancing gliomas [39].

#### **4. Conclusion**

Noncanonical IDH mutations are observed in only a limited number of all gliomas and are exceedingly rare among glioblastomas. It is unclear if tumors with these mutations are associated with more favorable outcome compared to canonical IDH mutants. Further study of the natural history of noncanonical IDH-mutant cancerous gliomas and analysis of the treatment effect of IDH mutation-specific targeted therapy is needed in the future.

#### **5. Recommended articles**

The recommended articles are [1, 4, 5, 15, 24, 29, 31, 32, 34, 39].

#### **Abbreviations**


*Glioblastoma - Current Evidence*

### **Author details**

Tariq D. Al-Saadi1,2 and Roberto J. Diaz1,2\*

1 Faculty of Medicine, McGill University, Montreal, Canada

2 Department of Neurology and Neurosurgery, Montreal Neurological Institute, McGill University, Montreal, Canada

\*Address all correspondence to: roberto.diaz@mcgill.ca

© 2022 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

*Noncanonical (Non-R132H) IDH-Mutated Gliomas DOI: http://dx.doi.org/10.5772/intechopen.105469*

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#### **Chapter 7**

## New Approaches in the Treatment of Glioblastoma Multiforme

*Lee Roy Morgan, Branko S. Jursic, Marcus Ware and Roy S. Weiner*

#### **Abstract**

Central nervous system (CNS) malignancies are rare, but commonly fatal and glioblastoma (GBM) is the most common of the primary brain tumors. In contrast to metastatic malignancies involving the CNS, which have external blood supplies that develop when the malignant cells penetrate the blood-brain-barrier (BBB), GBM generates its own intracerebral neovascular support system. Thus, the therapeutic issues as discussed herein review the development of drugs and therapeutics that will penetrate the BBB and are cytotoxic *to* GBM and other brain tumors. Since GBM is a CNS malignancy with minimal effective therapeutic options available, designing drugs and therapeutics as treatment for this malignancy that penetrate, but do not disrupt the BBB is the goal of this chapter. 4-Demethylcholesteryl-4-penclomedine (DM-CHOC-PEN) was designed and developed because of its lipophilic properties that *would* potentiate crossing the BBB and penetrate brain tumors. The drug has now completed Phase I/II clinical trial in humans with primary brain malignancies *demonstrating* objective responses in GBM. In addition, preliminary experiences with naturally occurring polyphenols—curcumin, quercetin, catechins and phloretin and derivatives—are reviewed as potential naturally occurring anti-glioblastoma agents.

**Keywords:** temozolamide (TMZ), glioblastoma multiforme (GBM), recurrence, radiosurgery, chemotherapy, 4 demethyl-4-cholesteryloxycarbonylpenclomedine, DM-CHOC-PEN, and multimodality treatment

#### **1. Introduction**

Approximately 48% of all primary malignant brain tumors are glioblastoma multiforme (GBM), and more than 10,000 people will succumb to the disease in the US each year alone (1). The 5-year relative survival rate for these patients with GBM increased only from 23%, as reported in the mid-1970s, to 36% in the early 2000s [1, 2]. Adding to the complexity of the disease is the cancer's ability to rapidly mutate, so even in different locations in the brain of the same patient, GBMs encompass a mosaic of cancer cell types, posing a major challenge for tumor-targeted therapy [3]. Thus, despite the advancements in the management and treatments for malignancies which we review here, the prognosis for long term survival for glioblastoma (GBM) continue to be dismal [4].

#### **2. History of the disease**

For metastatic cancers involving the brain there are cancer-associated-breaks and related neovascular channels in the BBB that allow drug penetration [4]. However, GBMs commonly lack facilitating neovascular changes in the BBB and must rely on drug lipophilicity and/or target transport mechanisms for anti-cancer agents to penetrate the BBB. GBM responses to the present therapies available are dismal and new therapies that penetrate the BBB are needed. Classically GBMs induce their own intracerebral neovascular blood supply within the brain that supplies the tumor mass with blood and nutrients—no extra-BBB blood supplies are involved; thus, the principle issue is penetrating the BBB [2, 5].

The major goal of this article is to initiate new ideas in the management of GBM, as well as other types of CNS malignancy. The core therapeutic challenge is to obtain long term objective responses through mechanisms involving drug penetration of the brain *via* the BBB and utilizing the changes in the chemistry of GBM malignancies.

In the treatment of GBMs, for drug and therapy modalities to be effective, there must be small/diffuse vascular accesses/openings or breaks (surgery sites), or lipophilic target pathways through the BBB secondary to interactions with a receptor or transport mechanism for penetration of the brain and CNS tumor masses [6].

Needless to mention, many treatment approaches that should be useful therapies for GBM also possess toxicities secondary to particle size and/or interaction with inappropriate sites—locally or distant—and unable to reach the tumor masses, and are not employed.

GBM has not been associated with smoking or any other lethal factors. The tumor remains the most common and lethal form of CNS brain cancers and one of the most difficult to manage.

In summary, since GBMs do not induce neoplastic blood support systems, drug and immune tumor targets, immune therapies do not easily penetrate the BBB and GBMs have not responded well to systemic therapies and new therapies should be aggressively evaluated [1, 2, 7, 8].

#### **3. Current drug therapies**

The O6 -methylguanine-DNA-methyltransferase (MGMT) gene encodes for an important DNA repair protein which acts by removing alkyl products from the O6 position on guanine. A so-called "suicide enzyme," following removal of the alkyl groups, the newly alkylated MGMT protein, is then marked for degradation by ubiquitination [8]. Proper functioning of the gene is important for maintaining cell integrity. Epigenetic silencing of the MGMT gene by methylation of the CpG islands of the promoter region has been shown to correlate with loss of gene transcription and protein expression [9, 10]. Loss of expression of the MGMT protein results in decreased DNA repair and retention of alkyl groups, thereby allowing alkylating agents such as carmustine (BCNU), lomustine (CCNU), and temozolomide to have greater efficacy in patients whose tumors exhibit hypermethylation of the MGMT promoter, reducing the MGMT protein concentration [10–13].

#### **3.1 Temozolamide**

Temozolamide (Temadar, TMZ) (**Figure 1**) continues to be the standard therapy +/− radiation for GBMs. However, the benefit of the therapy has been less than

*New Approaches in the Treatment of Glioblastoma Multiforme DOI: http://dx.doi.org/10.5772/intechopen.105886*

**Figure 1.** *Temozolamide (Temadar, TMZ).*

**Figure 2.** *Mechanism of action for TMZ.*

desirable since methylated *MGMT*- GBMs are most sensitive, as well uncommon [11–13]. Once TMZ passes through the BBB its mechanism of action is as follows:

TMZ is quickly and almost completely absorbed from the gut, and readily penetrates the blood– brain barrier and brain. The concentration of drug in the cerebrospinal fluid is approximately 30% of the concentration in the blood plasma. Intake with food decreases maximal plasma concentrations by 33%. TMZ is a prodrug; it is hydrolyzed at physiological pH to 3-methyl-(triazen-1) imidazole-4-carboxamide (MTIC), which further splits into monomethyl hydrazine—likely the active methylating agent—and 5-aminoimidazole-4-carboxamide (AIC) [13].

TMZ also induces breaks in the BBB and transforms several tumor marker receptors [13]. The therapeutic benefit of temozolomide depends on its ability to alkylate/ methylate DNA, which most often occurs at the N7 or O6 positions of guanine residues *via* the methyl hydrazine metabolite (**Figure 2**).

The time of day that the drug is administered may be of importance. Drug administered early in the AM appears to be more active than when administered in the evening. Since the drug is lipophilic and morning meals are commonly high in lipids may be a possible explanation.

#### **4. Core therapeutic challenges to obtaining long term objective tumor responses**

There are numerous challenges to be considered when designing new drugs as therapy for primary CNS malignancies.


Due to the Warburg-associated inductive effects present, cancer cells utilize glucosamine in contrast to glucose in the Krebs cycle for energy [3, 4, 14]. Although breaks in the BBB similar to those seen in metastatic cancers involving the CNS are not observed in the brains with GBM present, microneovascular support is present in the GBM associated para-cerebral environment.

There are new reports regarding tumor-target marker agents that are demonstrating activity against target-negative GBMs, however, more new agents that do not require a tumor-target marker for activity are needed [1]. Drug design needs to take advantage of natural target mechanisms *via* the BBB [3].

In this chapter we discuss several interesting non-tumor target designed agents [2].

#### **4.1 Designing agents to diffuse through the BBB**

4-Demethylcholesteryl penclomedine (DM-CHOC-PEN) (**Figure 3**.) was designed and developed because of its lipophilic properties that was anticipated to potentiate crossing the BBB and penetrating brain tumors [3]. The basic nucleus penclomedine was developed at the NCI- Southern Research Institute as treatment for brain tumors, but was withdrawn from clinical trials because of CNS toxicities (seizures) [14, 15]. The cholesteryl ester was added to the penclomedine nucleus at DEKK-TEC (see below) to increase lipophilicity [1].


#### **Figure 3.**

*DM-CHOC-PEN and functional moieties. [3, 14–16].*

#### **4.2 DM-CHOC-PEN—Mechanism of action**

During Phase I clinical pharmacodynamics studies when DM-CHOC-PEN was administrated IV, the drug was identified associated with erythrocytes (~50%), which is considered to be the mechanism by which it enters the cerebral circulation (**Figure 3**) and, therefore, available for transport into the tumor bed and the cancer cells resident therein [1, 4, 6]. The drug has been identified in intracranial metastatic NSCLC tissue [1, ].

Glucosamine is a component of the mucopolysaccharides that involved in the chemistry of red blood cell (RBCs) surface membranes and DM-CHOC-PEN has an affinity for glucosamine (**Figure 4**) [4]. Association of DM-CHOC-PEN with

**Figure 4.**

*DM-CHOC-PEN transport into CNS with glucosamine on RBC surfaces.*

#### **Figure 5.**

*DM-CHOC-PEN's transport into cells—Similarity with glutamine—Similarity with glutamine. NAD = pyridine nucleotides, KYN = kynurenine, and TRY = tryptophan.*

glucosamine allows the drug to form complexes with the surface of RBCs and be transported into the brain (**Figure 4**).

After transportation through the BBB and into the brain, DM-CHOC-PEN is transported into cancer cells with glutamine because of similarities with that structure (**Figure 5)**.

**Figure 6** represents a possible complex between DM-CHOC-PEN and glutamine that can occur after the former penetrates through the BBB into the brain and transported into GBM cells. Cancer cells, especially GBM, utilize glutamine as a source of C & H for ATP synthesis [4]. The Warburg effect is present in the GBM cells, thus glucose is not utilized for ATP synthesis [4].

**Figure 7** continues to be the best explanation of how DM-CHOC-PEN penetrates the BBB and intracerebral GBMs [2–6]. This mechanism was proposed by our group several years ago and continues to be a working model [2–6].

**Table 1** reviews the results reported during Phase I/II clinical trial with DM-CHOC-PEN in primary brain tumors in adults [5, 17]. Unfortunately, as noted in **Table 1**, DM-CHOC-PEN is not active in all GBMs treated to date [5, 17].

#### **Figure 6.**

*Complex of DM-CHOC-PEN with glutamine.*

*Overview—Mechanism of action for DM-CHOC-PEN [2–6].*

*New Approaches in the Treatment of Glioblastoma Multiforme DOI: http://dx.doi.org/10.5772/intechopen.105886*


*\*\*\*Method of Treatment—DM-CHOC-PEN (mg/m2 ) was infused IV over 4 hr. every 21 days to each patient—aged 37–78 y/o [5, 17]; NED—no evidence of disease [5, 17].*

#### **Table 1.**

*Primary brain tumor response to DM-CHOC-PEN therapy by cancer type-during phase I/II trials.*

During the trials, tumor tissue from several patients that received the drug were obtained and analyzed. Adducts were identified that support the DNA sites that are alkylated by DM-CHOC- PEN and do not involve O6 -guanine sites, thus DM-CHOC-PEN should be active in most types of GBMs (**Figure 8**).

#### **4.3 Immunotherapy targets**

The principle challenge to the treatment of GBM, as exists for all tumors involving the CNS, is the difficulty to penetrate the blood brain barrier (BBB) and deliver drugs into the CNS and GBM [1]. In GBM, the BBB is weakened, allowing immune cells from the periphery to penetrate the CNS. However, GBM tends to selectively attract or turn immune cells that infiltrate the tumor into immune suppressive cells which lack anti-cancer activity [1].

Most immunotherapies target the reactivation of effector T-cells, which attack and eliminate cancer cells. But, in GBM, the effector T-cell infiltration is very low, secondary to the above inhibitory immune suppressive properties, and there is an abundance of immunosuppressive myeloid cells and a low concentration of cytotoxic cells [1]. Thus, developing immune modulators that prevent impairment of cytotoxic lymphocytes is of potential importance and could be useful alone or with cytotoxic agents [1].

#### **4.4 Phenolic anti-glioblastoma compounds**

Medical application of phenolic compounds is well documented through decades [18]. Current research trends are exploring the senotherapeutic activity of these agents [19–22]. The elevation of the presence of the senescent cells seems to be the central part of aging and age- related diseases including cancer [23]. There are increasing numbers of reports referring to the use of plant extracts that are phenolic compounds in ant-glioblastoma studies [24, 25].

Considering that targeted therapy for glioblastoma has had promising results *in vitro* monolayer cultures, the results from preclinical and clinical trials has been disappointing partly due to the poor blood brain barrier penetration. There currently is more emphasis on application of natural phenols that are able to penetrate the BBB as alternatives for glioblastoma treatment [26, 27].

Anti-glioblastoma activity has been investigated in depth for several phenols curcumin, quercetin, catechins, and phloretin, to name a few [28–31]. In two-dimensional cell line tests, the above demonstrated that their IC50 values were ~ 50 μM concentration [32]. In addition, less than 2% of low molecular weight organic molecules crossed the BBB. For some phenolic compounds that cross the BBB a positive anti-cancer effect was observed [33]. However, the presence of phenolic compounds in the brain has been confirmed in only a few reports.

**Figure 9** reviews natural phenols that have been extracted from plant purified and tested for anti-glioblastoma activity; majority of the phenols were glycosylated [34]. It has been well demonstrated that glycosylated organic compounds easily cross BBB [34, 35]. A perfect example of a glycosylated phenol that crosses the blood-brain barrier is curcumin oligosaccharide.

The IC50 value for curcumin after 24 hr. in a U87 cell culture was 10 μM and 13 μM for cultures with T98 cells [34]. However, in pre-clinical mice studies there was no detectable amount of curcumin in the brain or in T98 GBM cells after its intraperitoneal injection (IP). On the other hand, when curcumin gluco-oligosaccharide was injected I.V. to mice, 18 ng of curcumin per 1 g of brain tissue was determined. In addition, 5 days after the IP injection of the oligosaccharide into C57BL mice bearing intracerebral brain tumors, complete responses were observed [36].

This suggests that two-dimensional cell test results for anti-glioblastoma oligosaccharide conjugates can be translated to animal models. With this in mind phenolic

**Figure 9.**

*Structures of naturally occurring anti-glioblastoma phenols.*

*New Approaches in the Treatment of Glioblastoma Multiforme DOI: http://dx.doi.org/10.5772/intechopen.105886*


*GBM cell line LN229 growing in culture was employed as the test system. Test compounds were evaluated in the 100 nanomolar—100 μM conc. Ranges VLR = violations Lipinski's 5 rule; CV = Cell viability (% of control) mean ± SD at 25 μM; VLR = violations Lipinski's 5 rule; BBB Score = Blood-brain barrier score [38]; CNS-MPO = central nervous system—multiparameter optimization [39].\* Fenofibric acid (2 microgm/mL) was used as a standard and demonstrated no activity in culture.*

#### **Table 2.**

*Comparison of cell (LN229) viability and compute brain penetration ability for fenofibric acid phenols HR49, HR51, and HR54\* .*

derivatives of fenofibric acid are being evaluated in clinical trials to document their activity as anti-GBM agents [37].

A number of phenolic fenofibric acid derivative were synthesized and tested against the glioblastoma LN229 cell lines. There were numerous fenofibric acid derivatives that had IC50 values between 1 and 10 μM (see below).

Several of the derivatives are listed in **Table 2**. According to computational studies, a majority of these compounds have high lipophilicity (logP >5), but their probability of crossing the BBB was below 50%. Studies *in vivo* (mice) indicated that the phenols did cross the BBB and traces of the compounds were detected in the brain and GBM tissue [37]. The fenofibric acid phenols are believed to inhibit GBM proliferation *via* reducing metabolic activity (ATP production), resulting in apoptosis of GBM with cell death. This is very similar to the experiments that were performed with curcumin [32].

Thus, the development of prodrug glycosylated fenofibric phenols that inhibit GBM cellular replication appear to be a promising viable approach to penetrating the BBB and cytotoxic therapy *vs* GBM.

#### **5. Conclusion**

An attempt to review the chemistry, neuropharmacology and preliminary results—*in vitro* and *in vivo—*has been made. DM-CHOC-PEN has been studied in depth, as therapy for both metastatic and primary malignancies involving the CNS. The latter is a bi-functional alkylating agent as discussed in this paper. However, minimal information is available regarding cytotoxic mechanisms of action for the polyphenolic structures described herein. The positive responses observed and reported are support for continued studies with the poly phenols as well as initiation of a Phase III clinical trial with DM-CHOC-PEN in GBM.

#### **Acknowledgements**

Supported by—NCI/SBIR—R43/44CA132257 & NIH NIGMS 1 U54 GM104940.

### **Abbreviations**


### **Author details**

Lee Roy Morgan1 \*, Branko Jursic2 , Marcus Ware3 and Roy S. Weiner4

1 DEKK-TEC, Inc., New Orleans, Louisiana, USA

2 Department of Chemistry, University of New Orleans, New Orleans, Louisiana, USA

3 Department of Oncology Neurosurgery, Ochsner Medical Center, New Orleans, Louisiana, USA

4 Department of Medicine, Section of Hematology and Medical Oncology, Tulane Medical Center, New Orleans, Louisiana, USA

\*Address all correspondence to: lrm1579@aol.com

© 2022 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

*New Approaches in the Treatment of Glioblastoma Multiforme DOI: http://dx.doi.org/10.5772/intechopen.105886*

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