Only subsets of patients benefit from vaccination/oncolytic virotherapy. In clinical trials, immunotherapies/oncolytic viruses are assessed concurrently with or after standard therapy. Importantly, arguing against its putative positive immunomodulatory role, standard therapy induces systemic immunosuppression and long-lasting severe lymphopenia [178–187], and may interfere with the immunotherapy/oncolytic virotherapy efficacy, which is critically dependent on the activity of the host's own immune cells [10,159,188–197]. In support of this, recent studies have reported that high

blood CD3+/CD4+ T cells counts [159] and tumor-infiltrating lymphocyte density [188] were correlated with better overall survival in glioblastoma patients receiving dendritic cell vaccination, while adjuvant TMZ hampered a CD8+ T cell count increase and the generation of CD8+ T cell-associated antitumor memory promoted by dendritic cell vaccination [198]. Standard therapy differentially affects the immune system of each patient [198–200], and patients with less severe standard therapy-induced immune suppression might derive more benefit from immunotherapy/oncolytic virotherapy than severely immunosuppressed patients. Detailed blood/tumor immunophenotyping should be incorporated in immunotherapy/oncolytic virotherapy trials to correlate immune cell subsets (counts, phenotype), immune responses to therapy and survival of patients to find immune-related predictive/prognostic markers.

## **8. Conclusions**

Since 2003, only two chemotherapeutic agents have been approved for the treatment of newly diagnosed glioblastoma: BCNU/carmustine and temozolomide. In 2015, tumor-treating alternating electric fields (TTFields) generated by the NovoTTF-100A device was approved by the FDA as a new glioblastoma treatment modality concurrently with standard therapy. However, more randomized controlled studies with long follow-up are required to assess the real clinical efficiency of the addition of TTFields to standard therapy in terms of overall survival and the 2-, 3- and 5-year survival rates. To date, no cytotoxic chemotherapeutic agent, antibody, molecularly targeted drug or combinations of small molecule inhibitors have been demonstrated to be more efficient than TMZ or to increase survival when combined with standard therapy. The addition of vaccines or oncolytic virotherapy to standard therapy has markedly improved the survival of subsets of patients in early-phase clinical trials. Large controlled trials are required to prove the efficacy of next-generation immunotherapeutics and oncolytic vectors. Since immunotherapy efficacy critically depends on the activity of the host's own immune cells, blood cell counts and immunophenotyping may potentially help find immune-related predictive/prognostic markers in immunotherapy/oncolytic virotherapy trials.

**Funding:** The work was financially supported by the Russian Foundation for Basic Research (RFBR), Grant No. 18-29-01009.

**Conflicts of Interest:** The authors declare no conflict of interest.

## **References**

© 2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

## *Review* **Glioblastoma in Elderly Patients: Current Management and Future Perspectives**

**Giuseppe Minniti 1,\* , Giuseppe Lombardi 2 and Sergio Paolini 3**

Received: 31 January 2019; Accepted: 5 March 2019; Published: 8 March 2019

**Abstract:** The incidence of glioblastoma (GBM) in the elderly population is slowly increasing in Western countries. Current management includes surgery, radiation therapy (RT) and chemotherapy; however, survival is significantly worse than that observed in younger patients and the optimal treatment in terms of efficacy and safety remains a matter of debate. Surgical resection is often employed as initial treatment for elderly patients with GBM, although the survival benefit is modest. Better survival has been reported in elderly patients treated with RT compared with those receiving supportive care alone, with similar survival outcome for patients undergoing standard RT (60 Gy over 6 weeks) and hypofractionated RT (25–40 Gy in 5–15 daily fractions). Temozolomide, an alkylating agent, may represent an effective and safe therapy in patients with promoter methylation of O6-methylguanine-DNA-methyltransferase (*MGMT*) gene which is predictor of responsiveness to alkylating agents. An abbreviated course of RT, 40 Gy in 15 daily fractions in combination with adjuvant and concomitant temozolomide has emerged as an effective treatment for patients aged 65 years old or over with GBM. Results of the National Cancer Institute of Canada Clinical Trials Group (NCIC CTG CE6) and European Organization for Research and Treatment of Cancer (EORTC 26062/22061) randomized study of short-course RT with or without concurrent and adjuvant temozolomide have demonstrated a significant improvement in progression-free survival and overall survival for patients receiving RT and temozolomide over RT alone, without impairing either quality of life or functional status. Although combined chemoradiation has become the recommended treatment in fit elderly patients with GBM, several questions remain unanswered, including the survival impact of chemoradiation in patients with impaired neurological status, advanced age (>75–80 years old), or for those with severe comorbidities. In addition, the efficacy and safety of alternative therapeutic approaches according to the methylation status of the O6-methylguanine-DNA methyl-transferase (*MGMT*) gene promoter need to be explored in future trials.

**Keywords:** glioblastoma; elderly; surgery; radiotherapy; chemotherapy; temozolomide

## **1.  Glioblastoma (GBM) is the most frequent histology and accounts for more than 50% of gliomas in all age groups, with an incidence rate among elderly patients of 70 years and older of 17.5 per 100,000 person-years, and a relative risk of 3–4 times compared with young adults [1–3]. Considering that the population of 65 years or older is expected to increase in the next two decades in USA, Canada, Australia, and Europe, this age group will account for the majority of GBM cases in these nations, representing an important aspect of public health.

Based on the results of the EORTC-NCIC CTG phase III study showing a significant improvement in the median survival for patients aged 18–70 years who received chemoradiation over radiation therapy (RT) alone from 12.1 to 14.6 months, and an improvement in 2-year survival from 10% to 26%, respectively, the standard of care for adult patients with GBM is represented by post-operative standard RT (60 Gy in 30 fractions) with concurrent and adjuvant temozolomide [4,5]. However, the majority of elderly patients with GBM are less likely to receive standard chemoradiation because aggressive combined approaches are associated with lower survival benefit and increased toxicity.

Elderly patients with GBM have historically been treated with standard or hypofractionated RT, with a reported median survival in the range of 5 to 9 months [6–17]. For elderly patients with O6-methylguanine-DNA methyl-transferase (*MGMT*) gene promoter methylation, the alkylating agent temozolomide has emerged as an effective treatment option associated with a survival benefit [18–20]. Published results from the EORTC (26981-22981)/NCIC CTG (CE.3) randomized study of short-course RT with or without concurrent and adjuvant temozolomide have made a significant contribution to the management of GBM [21] showing that the addition of temozolomide to short-course RT in patients 65 years of age or older with newly diagnosed GBM resulted in significantly longer survival compared to short-course RT alone. Although this regimen is likely to become the new standard of care in the elderly population with GBM, questions if all older patients may receive combined chemoradiation regardless of advanced age (>75–80 years old), impaired neurological status, presence of comorbidities, or molecular profiling of the tumor remain open.

The purpose of this review is to summarize the published literature on the efficacy of RT and chemotherapy given alone or in combination in elderly patients with GBM, and to address important issues such as the importance of molecular profiling in predicting response to treatments, the impact of treatments on quality of life and neurocognitive outcomes, and future research priorities for this population.

## **2. Methods**

There is no generally agreed criterion for definition of "older people". A cut-off of over 60 or 65 years is often used (http://www.who.int/healthinfo/survey/ageingdefnolder/en/). Most developed world countries have accepted the chronological age of 65 years as a definition of "elderly" or older person; however, in the developed countries, the most relevant geriatric syndromes (e.g., insomnia, urinary incontinence, severe hearing/vision problem, functional decline, fall and depressive disorder) are most common over 70–75 years. In order to include all published studies reporting on older patients with GBM, we have used an age cutoff of 60 years old for defining older patients in the current research.

A literature search was conducted in MEDLINE PubMed evaluating older people with GBM. The search focused on randomized, prospective and retrospective studies published in English. The literature search was performed using a combination of medical subject headings (MeSH) "glioblastoma" and free text terms ("radiation therapy" or "hypofractionated radiotherapy" or "chemotherapy" or "chemoradiation" or "elderly"). We included relevant studies published from 1990 to 2017. Studies published in languages other than English or not involving human subjects were not reviewed. A total of 178 potentially relevant studies were identified, including 19 prospective/randomized studies and 159 retrospective studies. The results of the literature research were used and included if appropriate.

#### **3. Overview of Treatments**

#### *3.1. Surgery*

Surgical resection is the first step in treating patients with GBM. The goals of the surgical procedure include histologic diagnosis, relief of the tumor mass effect, safe tumor cytoreduction, and possibly prolong patient survival. Although several reports have found that extensive surgical resection is

associated with longer survival [22–32], aggressive surgery is still a controversial issue in neurosurgery. Simpson et al. [22] reviewed the impact of the extent of surgery on the survival outcome in 645 patients with GBM who were enrolled in three consecutive randomized Radiation Therapy Oncology Group (RTOG) trials. Patients undergoing gross total resection had a significant longer median survival than those receiving a biopsy only (11.3 vs. 6.6 months; *p* < 0.001); notably, a significant difference in median survival times was also found for partial resection compared with biopsy only treatment (10.4 vs. 6.6 months; *p* < 0.01). In a series of 788 patients who underwent surgery for a malignant glioma between 1997 and 2001 in North America, Laws et al. [23] showed that total/subtotal surgical resection was an independent favorable prognostic factor compared with biopsy only (*p* < 0.0001), with no difference between older and younger patients. In another series of 1229 patients aged less than 80 years with histologically verified GBM undergoing surgery at the University of Texas MD Anderson Cancer Center, Li et al. [32] reported a median survival time of 15.2 months after complete resection and 9.8 months after incomplete resection (*p* < 0.001). This survival advantage was achieved without an increased risk of overall or neurological postoperative deficits even after correcting for prognostic factors including age, Karnofsky Performance Scale (KPS) score, and preoperative contrast-enhancing tumor volume. The favorable impact of complete resection on survival has been confirmed in published series reporting the use of modern surgical techniques, such as intraoperative magnetic resonance imaging, intraoperative ultrasonography, and fluorescence-guided surgery [28,29,33]. In a series of 243 patients enrolled in a phase III trial assessing the efficacy of fluorescence-guided surgery with 5-aminolevulinic acid for resection of malignant gliomas, Stummer et al. [29] showed that patients who underwent complete resection survived longer than those who had incomplete resection (16.7 vs. 11.8 months, *p* < 0.0001), even in elderly patients older than 60 years of age with GBM.

A few studies have evaluated the efficacy of surgery in elderly patients with GBM [34–40]. In a systematic review and meta-analysis of biopsy versus partial versus gross total resection in patients older than 60 years of age with high-grade glioma, Almenawer et al. [37] have compared overall survival, KPS, progression-free survival, mortality, and morbidity amongst 12607 patients who were included in 34 studies. The overall survival was 5.71 months (95% CI 5.04–6.36) in patients undergoing biopsy, 8.68 months (95% CI 7.87–9.48) in those having subtotal resection, and 14.04 months (95% CI 12.8–15.2) in those subjected to gross total resection. For the entire population, patients undergoing overall resection (of any extent) had a significant benefit compared with those having biopsy, with mean difference in overall survival of 3.88 months (95% CI 2.14–5.62, *p* < 0.001). Mean difference in postoperative KPS was 10.4 (95% CI 6.58–14.22, *p* < 0.001) and mean difference in progression-free survival was 2.44 months (95% CI 1.45–3.43, *p* < 0.001). Overall, the analysis showed longer survival time, delayed tumor progression rate, and improved functional recovery with decreasing trends of mortality and morbidity rates in the order of biopsy, sub-total resection, and complete resection, suggesting a progressive improvement in clinical outcomes with greater degrees of resection. In a small randomized study of 30 patients older than 65 years with malignant glioma who received stereotactic biopsy or surgical resection, Vuorinen et al. [35] reported median survival times of 171 and 85 days after surgical resection or biopsy, respectively (*p* = 0.035). Similar results have been reported in other retrospective series showing significantly improved survival in patients with GBM receiving subtotal/complete resection compared with those undergoing stereotactic biopsy [36–40].

Biopsy, which has limited mortality and serious morbidity in the range of 1–5%, is usually performed to assess histological and molecular characteristics of the tumor when surgical resection carries high risks [41–44]. Tumor tissue is in fact fundamental to assess the molecular profile of GBMs and consequently to tailor the appropriate treatment. In this contest biopsy can be avoided only when clinical and radiological data provide an accurate diagnosis of GBM and results will not affect treatment choices.

Overall, findings from these studies support the general principle of considering maximal degrees of tumor removal when the operative option is indicated, regardless of age. The main limitation of these studies is a lack of detail about surgical complications, functional recovery time, and neurocognitive outcome following variable levels of resections. Preventing new permanent neurological deficits and maintaining good quality of life are essential factors for guiding surgical management of these patients. In absence of randomized studies, in current clinical practice the optimal surgical approach in elderly patients should be individually based on the carefully evaluation of known established safety measures, as those included in the neurologic assessment in neuro-oncology (NANO) scale [45] and in geriatric assessment models [46].

#### *3.2. Radiotherapy*

Postoperative RT, either standard RT or abbreviated courses of RT, has been historically employed for elderly patients with GBM [6–17]. A summary of randomized controlled trials addressing the efficacy and safety of RT in the elderly population with GBM is shown in Table 1 [14,15,17,19,20,40].

The superiority of RT (50 Gy in 28 daily fractions) over best supportive care has been demonstrated in a French multi-institutional randomized trial of 85 elderly patients with GBM aged 70 years or older with a KPS score of 70 or higher [47]. Median overall survival and progression-free survival times were 29.1 and 14.9 weeks for patients receiving RT plus supportive care and 16.9 and 5.4 weeks for those receiving supportive care alone, respectively. Notably, RT did not cause further deterioration in the KPS, health-related quality of life and cognitive functions compared with supportive care. The efficacy of RT versus supportive care alone has been demonstrated in other few studies [6,8,10–12,14,16,48]. Using the Surveillance, Epidemiology, and End Results (SEER) registry (1988–2004) as data source of 10987 patients with GBM aged 70 years or older, Scott and colleagues [41] showed that the overall survival time was significantly improved by RT after adjusting for surgery, tumor size, gender, ethnicity, and age at diagnosis. In another retrospective series of 202 patients with GBM treated between 1990 and 2000 at Leiden University, Marijnen et al. [16] reported a significant longer survival of 10.6 months in patients treated with RT compared with 1.9 months in non-irradiated patients (*p* < 0.0001), with no significant survival difference between elderly and young patients.

Few randomized studies have addressed the efficacy and safety of either radical RT or abbreviated courses of RT in elderly patients with GBM [15,17,19] (Table 1). In a randomized trial of 100 patients with GBM aged 60 years or older who received postoperative standard RT or short-course RT (40 Gy in 15 fractions over 3 weeks), Roa et al. [15] showed no survival differences between the two groups. Median survival times and 6-month survival rates were 5.1 months and 44.7% for patients treated with standard RT and 5.6 months and 41.7% for those receiving short-course RT, respectively.

The Nordic randomized, phase III trial enrolled 291 patients older than 60 years of age with newly diagnosed GBM who were assigned to receive three different treatments: temozolomide (200 mg/m2 on days 1–5 of every 28 days for up to six cycles), hypofractionated RT (34 Gy given in 3.4 Gy fractions over two weeks), or standard RT (60 Gy given in 2 Gy fractions over 6 weeks) [19]. The efficacy of radiation treatments was similar between the two radiation groups. The median survival time was 7.5 months for patients treated with hypofractionated RT and 6.0 months for those receiving standard RT; however, in patients older than 70 years hypofractionated RT resulted in significantly longer survival than standard RT (7.0 months vs. 5.2 months, *p* = 0.02).

Roa et al. [17] conducted a randomized trial of 98 frail and/or elderly patients aged 65 years and older with GBM who were randomized to receive two different hypofractionated radiation schedules. Median overall survival and progression-free survival times were 7.9 and 4.2 months in patients who received 25 Gy in five daily fractions and 6.4 and 4.2 months in those subjected to 40 Gy in 15 daily fractions over three weeks, respectively (*p* = 0.9). Neurological outcome and quality of life were similar between the two groups at 4 weeks and 8 weeks after treatment.

The assessment of neurocognitive status and quality of life is of particular relevance in elderly patients with GBM (Table 1). In Roa et al. [15] trial comparing standard RT versus short-course RT, KPS scores varied markedly over time but were not significantly different between groups; notably, 20% of elderly patients receiving standard RT interrupted the treatment because of acute toxicity. In the

Nordic study, global health status and several functioning scales, including physical, role, emotional, social, functioning and cognitive (assessed by the European Organisation for Research and Treatment of Cancer (EORTC)) quality of life questionnaire Core 30 (QLQ-C30) did not change significantly between patients receiving standard or hypofractionated RT [19]. However, data need be interpreted with caution due to the low number of patients who completed questionnaires. A similar improvement of quality of life and performance status have been reported using hypofractionated schedules of 25–40 Gy in 5–15 daily fractions [6,8,9,14,17].

In summary, RT is associated with an improved survival in elderly patients with GBM with no significant detrimental effects on neurocognitive function and quality of life. Although standard RT may represent a feasible treatment option for elderly patients of 60–70 years old with a good performance status, results of randomized controlled studies comparing standard and hypofractionated radiation schedules clearly indicate that short-course RT should be recommended in elderly patients because it offers similar survival benefit and shortens the time of treatment. In addition, advanced radiation techniques, including intensity-modulated radiotherapy (IMRT), volumetric-modulated arc therapy (VMAT), and image-guided RT (IGRT), which allow the delivery of more precise radiation doses to the target while minimizing exposure of the surrounding normal brain tissues, should be routinely used in clinical practice to treat these patients with the objective to reduce the risk of neurocognitive deficits.

## *3.3. Chemotherapy*

Historically, the role of chemotherapy as initial treatment for elderly patients with GBM has been poorly investigated in the past, mainly because of the concern about the limited efficacy [49,50] and the severity of side effects of nitrosurea-based regimens [51,52]. Recently, the use of temozolomide as an alternative to RT in older patients with malignant gliomas has been addressed in prospective and randomized studies (Table 1) [18–20,53,54].

The German Neuro-oncology Working Group (NOA) phase 3 trial (NOA-08) has compared the efficacy and safety of RT to temozolomide in elderly patients with anaplastic astrocytoma or GBM; 373 patients aged 65 years and older with histologically confirmed tumors, and a KPS score ≥ 60, were randomly assigned to receive dose-dense temozolomide (one week on, one week off cycles) or standard RT [20]. Median survival times and one-year overall survival rates were 8.6 months and 34.4% for patients receiving temozolomide, and 9.6 months and 37.4% for those treated with standard RT, respectively, indicating that chemotherapy was non-inferior to standard RT. Similarly, median event-free survival times (progression or death as event) were not different: 3.3 months for patients treated with temozolomide and 4.7 months for those treated with standard RT. The major novelty of the study was the strong predictive role of *MGMT* promoter methylation status on event-free survival outcome. Event-free survival rates were significantly better in patients with *MGMT* methylated promoter than in those with *MGMT* unmethylated promoter, although this favorable impact was seen only in patients receiving temozolomide. For patients with a methylated *MGMT* promoter, event-free survival rates were better with temozolomide than RT, while the opposite was true for patients with an unmethylated *MGMT* promoter. Analysis of health-related quality of life scales showed no significant differences between the two groups; however, grade 2–4 adverse events were more frequent in patients receiving temozolomide.

**Table 1.** Selected prospective studies on radiotherapy or chemotherapy in older patients with glioblastoma.

708

+TMZ (200 mg/m2 1 week on/1 week off); Bev, bevacizumab.

In the Nordic trial, elderly patients receiving temozolomide had better survival outcome than those having RT. The median survival time was 8.3 months after temozolomide and 6.0 months after standard RT (HR 0.7, 95% CI 0.52–0.93, *p* = 0.01); however, with no significant differences between patients receiving standard RT and hypofractionated RT (6.0 months vs. 7.5 months, HR 0.82, 95% CI 0.63–1.06, *p* = 0.12) [19]. As for the NOA-8, a striking finding of the study was the predictive value of *MGMT* promoter methylation status. For patients receiving temozolomide, the median survival was 9.7 months in patients with *MGMT* promoter methylation and 6.8 months in those without methylation (*p* = 0.02). In contrast, *MGMT* methylation status did not affect the survival in patients having RT (methylated tumors, 8.2 months; unmethylated tumors, 6.8 months; *p* = 0.81). EORTC QLQ-C30 data evaluating global health status and cognitive functioning were generally better in patients receiving temozolomide than those having standard RT.

A French phase II trial including 70 patients aged 70 years and older with newly diagnosed GBM and a postoperative KPS score < 70, showed that temozolomide alone, given at doses of 150 to 200 mg/m2/die for 5 days every 4 weeks until disease progression, resulted in improved functional status and quality of life, with a substantial proportion of patients who became capable of self-care, especially those with an *MGMT* methylated tumor [18]. The median and 6-month survival rates were 31 weeks and 69.2% in patients with *MGMT* promoter methylation and 18.7 weeks and 28% in those without methylation (*p* = 0.03), respectively. The same ANOCEF French group has recently published the results of another phase II trial exploring the combination of temozolomide and bevacizumab in 66 patients aged 70 years and older with a KPS < 70 and histologically confirmed GBM [54]. With a median overall survival (OS) of 23.9 weeks, cognition and quality of life significantly improved over time during treatment, and 33% of patients became transiently capable of self-care. Grade 3 or 4 hematologic toxicity and high blood pressure occurred in 20% of patients. Other toxicities, including venous thromboembolism, cerebral hemorrhage, and intestinal perforation were less common (<5%).

In summary, temozolomide is an effective and tolerated treatment for elderly patients with GBM associated with a significant improvement of functional status and quality of life. Response to treatment is significantly associated with *MGMT* promoter methylation status. For patients with *MGMT* methylated tumors, temozolomide results in longer compared with standard RT; in contrast, there is no evidence of a survival benefit in patients with *MGMT* unmethylated tumor. In clinical practice, this means that postoperative temozolomide should be considered only in patients with *MGMT* promoter methylated tumors, whereas its use is not recommended in those with *MGMT* unmethylated tumors.

#### *3.4. Combined Chemoradiation*

The use of standard or hypofractionated RT in combination with concomitant and/or adjuvant TMZ has been evaluated in several studies [55–69]. Results from published prospective series are shown in Table 2 [21,56,57,60,64].

*Cancers* **2019**, *11*, 336

In a small prospective series of 32 patients aged 70 years and older with a good KPS receiving standard RT with adjuvant and concomitant temozolomide at Sant'Andrea Hospital (University of Rome Sapienza, Rome, Italy) the median survival time and one-year survival rates were 10.6 months and 37%, respectively [56]; grade 3 or 4 hematologic toxic effects occurred in 24% of patients. In another prospective study of 58 patients aged 65 years and older with GBM treated with standard RT and concomitant and adjuvant temozolomide, Brandes et al. [57] observed a median survival of 13.7 months. *MGMT* methylation status was an independent prognostic factor for survival; the two-year survival rates were 83% for patients with methylated tumors and 56% for those with unmethylated tumors. Grade 3 or 4 hematological toxicity occurred in 10% of patients and grade 3 neurocognitive deterioration in 25% of patients. A similar incidence of neurological toxicity up to one third of elderly patients with GBM treated standard chemoradiation has been reported in few series [58,59].

The efficacy and safety of hypofractionated RT with or without temozolomide has been recently evaluated by the intergroup EORTC 26062-22061/NCIC CTG (CE.3) randomized trial comparing an abbreviated course of RT (40 Gy in 15 fractions) plus concomitant and adjuvant temozolomide versus abbreviated RT alone in 562 patients older than 65 years old with newly diagnosed GBM [21]. The median survival time (9.3 vs. 7.6 months, *p* < 0.0001) and progression-free survival time (5.3 vs. 3.9 months, *p* < 0.0001) were significantly better in patients receiving combined chemoradiation over RT alone. *MGMT* promoter methylation status was a strong predictor for survival. Amongst 165 patients with *MGMT* methylated promoter, the overall survival was 13.5 months in patients receiving RT and temozolomide and 7.7 months in those receiving RT alone (*p* = 0.0001); in patients with *MGMT* unmethylated promoter, the respective overall survival times were 10.0 months and 7.9 months (*p* = 0.055). Quality of life analysis assessed by the EORTC QLQ-C30 questionnaire and the EORTC brain module (QLQ-BN20) showed that changes in global health status, functioning and symptom scales were similar in the two groups, although nausea and constipation were worse in patients receiving temozolomide.

A similar survival benefit has been reported in a phase 2 trial of 70 patients aged 70 years and older with newly diagnosed GBM treated with the same regimen at the University of Rome [64]. For the whole population, the median overall survival time and 1-year survival rates were 12.4 months and 58%, respectively. According to the *MGMT* promoter methylation status, the 1-year and 2-year survival rates were 81% and 20% in patients with *MGMT* methylated tumors, and 32% and 0% in those with *MGMT* unmethylated tumors, respectively (*p* = 0.0001). The treatment was well tolerated and resulted in a significant improvement or stability in global health, social functioning, and cognitive functioning scores between baseline and 6-month follow-up.

In summary, combined chemoradiation represents an effective therapeutic strategy for elderly patients with GBM. Standard RT and concomitant and adjuvant temozolomide remain a feasible treatment in patients aged less than 70 years with good KPS, although treatment-related neurotoxicity leading to serious disability and worsening of the quality of life represents a major concern. Based on the results of the EORTC 26062-22061/NCIC CTG (CE.3) randomized trial, elderly patients aged 70 year and older with *MGMT* promoter methylation who are considered eligible for combined modality treatment should be offered a short-course RT, 40 Gy in 15 fractions, in combination with concomitant and adjuvant temozolomide. Elderly patients not considered candidates for combined chemoradiation should be treated with short-course RT or temozolomide based on *MGMT* promoter methylation status [70].

#### **4. Future Perspectives**

Combined chemoradiation remains a matter of concern in frail patients presenting with functional deficits, multiple comorbidities and geriatric syndromes, like gait imbalance, malnutrition, delirium, and incontinence that make them more vulnerable to treatment-related toxicities [71–73]. Identification of frail, vulnerable, or fit patients is essential for making more appropriate treatment decisions for elderly patients. A comprehensive geriatric assessment which includes the evaluation of functional

status, cognitive function, nutritional status, comorbidities, polipharmacy, and socioeconomic status needs to be incorporated in future clinical trials with the aim of improving treatment outcome and reducing the risk of adverse events [74,75]. Several instruments may be used to assess the different domains of geriatric assessment, including performance status scales, daily activities, cognitive function, presence of comorbidities, psycological status, health and nutritional status, and socioeconomic status [46,74,76]. For daily clinical practice, several screening tests have been developed, including the abbreviated comprehensive geriatric assessment (aCGA) [77], the Groningen frailty indicator (GFI), as the G8 [78], and the vulnerable elders survey-13 (VES-13) [79] to help neurooncologists to guide cancer treatment decision-making and improve quality of life and functional independence of elderly patients with GBM. Based on geriatric assessment, patients with a higher frailty index score would receive less aggressive treatments; e.g., temozolomide or RT alone according to *MGMT* methylation promoter status.

Recently, experts from United States, Canada, and Europe have developed the Neurologic Assessment in Neuro-Oncology (NANO) scale which is an objective and simple tool for an accurate assessment of neurological function [45]. The NANO scale evaluates nine major domains of neurologic function that are most relevant to patients with supratentorial, infratentorial, and brainstem tumors, including gait, strength, upper extremity ataxia, sensation, visual fields, facial strength, language, level of consciousness, and behavior. It will provide an accurate neurological evaluation of elderly patients with GBM in both clinical trials and daily practice.

A novel treatment modality for patients with GBM is represented by the tumor-treating fields (TTFields) device (Optune®, Novocure Ltd., Novocure Inc, Israel) which is a portable, battery-operated device that generates TTFields. Results from a prospective phase 3 trial, EF-14, comparing TTFields plus temozolomide versus temozolomide alone after standard chemoradiation in patients with GBM has shown significant longer survival and clinical improvement in those having TTFields plus temozolomide [80]. Based on these results, Optune® has received FDA approval for adult patients with newly diagnosed supratentorial glioblastoma, in addition to standard postoperative chemotherapy, or as monotherapy for the treatment of recurrent GBM.

For patient of all ages, surgery, radiation and systemic therapies have been employed for recurrent tumors, although standards of care are not well defined. Currently, there are no prospective studies evaluating the management of older patients with recurrent GBM. Outside of the context of clinical trials, systemic therapy with either lomustine or bevacizumab may represent a feasible treatment option for fit patients with recurrent tumors. Randomized studies evaluating the efficacy of lomustine given alone or in combination with other agents have observed a median survival in the range of 8–10 months [81–83]. In a phase III trial of 437 patients assigned to receive lomustine plus bevacizumab or lomustine alone, Wick et al. [83] showed a similar survival between the two groups (9.1 months versus 8.6 months; *p* = 0.65). Grade 3 to 5 adverse events occurred in 63.6% of the patients in the combination group and 38.1% of the patients in the monotherapy group; this means that the use of lomustine should be carefully considered in older and frail patients at increased risk of toxicity. The antiangiogenic agent bevacizumab has been approved at recurrence in various countries, but not in the European Union. Although bevacizumab did not have superior efficacy compared with lomustine [82,83], it produces evident symptom relief and steroid-sparing effects. Future clinical trials need to evaluate the efficacy and safety of different treatment approaches in elderly patients with recurrent GBM and good functional status.

Although data from EORTC 26062-22061/NCIC CTG (CE.3) randomized trial support the combination of hypofractionated RT with temozolomide in patients older than 65 years of age, the survival benefit in patients with unmethylated tumors remains questionable. In addition, no trials have compared the survival and neurocognitive outcomes of combined chemoradiation versus temozolomide alone in patients with *MGMT* promoter methylation. Thus, future trials are needed to explore these important issues. Finally, molecular profiling contributes to identify prognostic subgroups of elderly patients with GBM who may benefit from new therapies. Although recent trials

have failed to demonstrate the efficacy of new agents in patients with GBM, including bevacizumab and cilengitide, the discovery of targeted agents and immunotherapy strategies which can translate to a survival benefit in patients with GBM remain an area of continued research.

## **5. Conclusions**

For elderly patients with newly diagnosed GBM, current management includes surgery, RT and chemotherapy; however, survival is significantly worse than that observed in younger patients. Standard RT with concomitant and adjuvant temozolomide, which represent the standard of care for newly diagnosed adult patients with GBM in good general and neurological condition, may be considered in selected fit patients aged between 65 and 70 years. Elderly patients aged 70 years and older who are considered eligible for combined modality treatment should receive a short-course RT with concomitant and adjuvant temozolomide up to 12 cycles. In the absence of comparative data between temozolomide alone and chemoradiation, elderly patients with *MGMT* promoter methylation may be considered for temozolomide alone, especially those presenting with functional impairment and geriatric syndromes. Temozolomide alone is not associated with survival advantages in elderly patients with unmethylated tumors; patients who are not considered eligible for combined chemoradiation should receive hypofractionated RT. Supportive and palliative care may represent an appropriate approach for frail patients with large or multifocal tumors and low KPS at an increased of treatment-related toxicity.

**Author Contributions:** G.M., G.L., and S.P. participated in article preparation, data analysis and wrote the manuscript. All authors have approved the final article.

**Funding:** This research received no external funding.

**Conflicts of Interest:** All authors declare that they have no competing interests.

## **References**

© 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

## *Review* **Allergic Signs in Glioma Pathology: Current Knowledge and Future Perspectives**

## **Massimo Costanza \* and Gaetano Finocchiaro**

Unit of Molecular Neuro-Oncology, Fondazione IRCCS Istituto Neurologico Carlo Besta, 20133 Milan, Italy<EMAIL_ADDRESS>
**\*** Correspondence<EMAIL_ADDRESS>Tel.: +39-02-2394-4654

Received: 21 February 2019; Accepted: 19 March 2019; Published: 22 March 2019

**Abstract:** Historically restrained to immune defense against parasite infections, allergic inflammation has been recently rediscovered to protect from a wide array of environmental triggers, such as xenobiotics and carcinogens, which can induce DNA damage and ultimately lead to cancer development. Moreover, cells and mediators typical of allergic responses can importantly modulate the tissue inflammatory milieu, which represents a crucial gatekeeper towards the acquisition of malignancy by cancer cells through immune escape. Numerous studies have described an inverse association between allergies and glioma development. Mast cells, key players of allergic reactions, have been recently found at increased numbers in glioblastoma multiforme (GBM), the most common and lethal primary brain tumor, and they have been implicated in GBM pathogenesis. In this review, we summarize epidemiological studies and discuss the main evidence highlighting a potential interplay between allergic responses, and glioma formation and progression. Last, we draw future lines of research for better clarification whether and through which mechanisms allergic inflammation might impact on gliomagenesis. The comprehension of the immune mechanisms favoring or counteracting tumor growth might open the path to novel immunotherapy approaches.

**Keywords:** allergy; glioma; GBM; IgE; IL-4; mast cells

## **1. Introduction**

Gliomas are the most common primary brain tumors of adults and are classified into different types and grades, based on histologic and molecular patterns [1]. Glioblastoma multiforme (GBM) accounts for approximately 70% of malignant gliomas and it is characterized by severe morbidity and high mortality [2]. Recent data suggest that GBMs arise from driver mutations of neural stem cells in the subventricular zone of the adult human brain [3]. Divergent genetic evolution may then be one factor underlying GBM progression [4]. The best-established risk factor for malignant gliomas is the exposure to ionizing radiation, however, a large body of literature has suggested that subjects with atopy have a diminished probability of developing gliomas [2]. The reason for this inverse association is still unknown. Type 2 immune reactions have long been ascribed to host defense against parasite infections, with allergic inflammation considered as an off-targeted side-effect of these protective responses [5]. Interestingly, recent lines of evidence have clearly shown that type 2 immunity exerts crucial functions in a complex array of responses, including venom detoxification as well as protection from hematophagous fluids, toxic xenobiotics and carcinogens [5]. These responses are driven by T helper (Th)2 cells, which produce mainly interleukin (IL)-4 and IL-13, that in turn promote immunoglobulin (Ig)E production by B cells, and IL-5, important mediator for eosinophil activation [6]. Allergic inflammation includes also IgG1 and components of innate immunity, such as mast cells, basophils, eosinophils, alternatively-activated macrophages and innate lymphoid cells [5].

The relationship between allergic inflammation and cancer has been the subject of several investigations, leading to opposite views regarding the impact of these responses on tumor growth. According to one current of thought, allergic reactions might foster tumor development by impairing Th1 and cytotoxic T cell functions, and by supporting myeloid cells with a suppressive phenotype [7]. Indeed, in a model of breast cancer, IL-4-producing Th2 cells have been shown to sustain metastasis by promoting effector functions of tumor-associated macrophages [8]. Conversely, according to an alternative viewpoint, allergic responses might be important for tumor-surveillance [5]. In a model of skin cancer induced by exposure to the carcinogen dimethylbenz[a]anthracene (DMBA), it was demonstrated that IL-4-deficient mice develop tumors of increased size and greater incidence compared to wild-type mice [9]. Moreover, sensitization to DMBA was associated with the establishment of a strong autoreactive IgE response that was protective against carcinogenesis [9]. A similarly puzzling scenario emerges from studies analyzing the contribution of mast cells to cancer, as they have been suggested as either protective or detrimental [10]. Most probably, depending on the specific neoplastic condition and the experimental model utilized, allergic inflammation can promote or counteract tumor development [10]. Very recently, the long controversy regarding the activity of eosinophils in tumor development has gained new insight from the study of Holland and colleagues [11,12]. In this work, it has been shown that in several solid tumors such as hepatocellular carcinoma, breast cancer and prostatic carcinoma, eosinophils can dampen tumor growth by inducing tumor cell-cytotoxicity. In particular, tumor-derived IL-33 was shown to promote chemokine CCL11-mediated eosinophil infiltration and degranulation [11,12].

In recent years, numerous studies have investigated the contribution of allergic components in the pathogenesis of gliomas and GBM. In this review, we summarize main epidemiological, histopathological and genetic studies that have suggested the involvement of allergic inflammation in glioma pathology and we discuss experimental approaches to further characterize in future the relationship between this specific arm of the immune system and brain tumorigenesis.

#### **2. Epidemiology of Gliomas and GBM in Allergic Subjects**

Most of the research on glioma and allergy has relied on epidemiological investigations, which have shown an inverse association between allergies and the risk of developing gliomas since the early 1990s [13]. In an international population-based case-control study performed in six different countries and including 1178 cases, it was reported that there is a reduced risk of 30–40% of developing gliomas among subjects with asthma, eczema and other allergic conditions [14]. In two subsequent case-control studies performed by Wiemels and colleagues on subjects enrolled in the San Francisco Bay Area, a significant inverse association for both self- and proxy-reported histories of allergic conditions with a diagnosis of adult glioma was shown [15,16]. A particularly decreased risk was found among subjects with allergies to pollen, dairy products and nuts [15]. Additionally, a strong association with diminished risk of developing gliomas has been detected in cases with late-onset (>12 years) respiratory allergies [16]. Another case-control study enrolling 489 cases of gliomas among three different hospitals in the United States (US), confirmed an inverse association between the risk of glioma and history of any allergy (e.g., eczema, allergy to insects, chemicals, etc.) [17]. Of note, in this work, people affected by both allergies and autoimmune diseases were at particularly low risk of developing glioma (odds ratio (OR) = 0.24, 95% CI, 0.14–0.42) [17]. In addition, a retrospective study has shown in two independent cohorts selected from Swedish twin registries that high-grade gliomas (grades III and IV) are inversely associated with allergic conditions that comprised of eczema, asthma, hay fever, and allergic rhinitis (hazard ratio, HR = 0.45 and 0.46, 95% CI) [18]. In a large, population-based case-control study in the UK, history of asthma, eczema, hay fever or other types of allergy was inversely correlated to the risk of developing glioma (OR = 0.63, 95% CI, 0.53–0.76) [19]. Association with allergies was not increased or decreased in grade I/II compared with grade III/IV tumors in this work [19]. Moreover, there was no evidence for a gradient of risk with the age of onset, time since onset or with the number of conditions presented [19]. A meta-analysis of both case-control and cohort studies published between 1979 and 2007 and including 3450 cases, has shown that the risk of glioma was decreased by 40% among subjects with a history of allergy, 30% diminished among those

with a history of eczema, and 30% reduced among those with a history of asthma [20]. Similar findings have been obtained in a large population-based case-control study published in 2007 and comprising 1527 cases of gliomas recruited in Denmark, Norway, Finland, Sweden and Southeast England [21]. This epidemiological trend has been confirmed also in another case-control study involving 366 cases of glioma recruited among the German population [22]. In an alternative approach, Il'yasova and colleagues have compared 388 glioma cases with three different groups of control—siblings, friends and clinic-based controls—to better evaluate the contribution of genotypic (for siblings) or environmental (for friends) effects on the relationship with allergic inflammation [23]. Allergies were always found significantly inversely associated with gliomas: ORs were 0.53 (95% CI, 0.15–1.84), 0.54 (95% CI, 0.28–1.07) and 0.34 (95% CI, 0.23–0.50) with siblings, friends and clinic-based controls, respectively [23]. A case-control study recruited 855 high-grade glioma patients from four different geographic regions of the US and belonging to five inherited glioma risk variants: 5p15.3 (*TERT*), 8q24.21 (*CCDC26/MLZE*), 9p21.3 (*CDKN2B*), 11q23.3 (*PHLDB1/DDX6*) and 20q13.3 (*RTEL1*) [24]. A significantly stronger inverse association of allergy history with glioma was detected among subjects who do not carry the glioma risk allele in the 9p21.3 region, while it was suggestively higher among those carrying the glioma risk allele in the 20q13.3 region [24]. In 2014, Cahoon and co-workers analyzed a cohort of 4.5 million male US veterans comprising 4383 cases of patients with a discharge diagnosis of malignant neoplasm of the brain, which included mainly gliomas, expected to cover 95% of cases, and some rare childhood tumors such as medulloblastoma. In this study, allergy/atopy of long latency (≥10 years) was associated with a reduced risk of developing brain cancer (rate ratio = 0.60, 95% CI, 0.43–0.83) [25]. Last, a recent multicenter case-control study carried out in four areas in France in 2004–2010 has explored the relationship between allergy (e.g., asthma, eczema, rhinitis/hay fever and other allergic conditions) and the risk of glioma in 273 glioma cases and 982 matched controls [26]. In addition to confirming previous findings, this work highlighted a dose-effect relationship between the number of allergic conditions and the inverse association with glioma risk and a stronger relationship in women [26]. To avoid the possibility that allergic inflammation might be somehow influenced by (or be the effect of) brain tumor itself, Schwartzbaum and colleagues have investigated germline polymorphisms as potential biomarkers of GBM susceptibility, selecting five single nucleotide polymorphisms (SNPs) strongly associated with asthma and allergic conditions [27]. Authors found that in subjects carrying three of these polymorphisms, located on the *IL-4 receptor α* (*IL4RA*) and *IL13* genes, the odds ratios for GBM were in the opposite direction with those for asthma [27]. Interestingly, pre-diagnostic serum levels of IL-4 and soluble IL-4RA have been later found inversely associated with gliomas and GBM in a nested case-control study including 487 glioma cases and 487 matched controls [28]. Of note, this association was present >20 years before glioma diagnosis [28]. Epidemiological data are summarized in Table 1.

**Table 1.** Epidemiological studies of allergy and risk of glioma.

Confidence interval (CI); relative risk (RR); odds ratio (OR); hazard ratio (HR). 1 Association of low-grade (I and II) glioma cases. 2 Association of high-grade (III and IV) glioma cases. 3 Brain tumors included rare childhood tumors, but mainly gliomas, expected to cover 95% of cases. Ref., reference.

## **3. Allergic Mediators in Glioma and GBM**

Much effort has been made to correlate the above described epidemiological observations with the prototypical biomarker of allergic inflammation, that is IgE. In 2004, Wiemels et al. found significantly lower IgE levels in glioma patients compared to controls (OR = 0.37, 95% CI, 0.22–0.64), with a more striking inverse association for IgE specific to food allergens (OR = 0.12, 95% CI, 0.04–0.41) [16]. However, a follow-up of this work by the same group has suggested that this inverse relationship is detectable only among cases receiving temozolomide [29]. Conversely, two later works have actually confirmed a relationship between serum IgE levels and gliomas. Indeed, a nested case-control study combining data from four prospective cohort studies have found a statistically significant inverse association between "borderline-elevated" total IgE levels (25–100 kU/L) and glioma (with 169 cases) (OR = 0.63, 95% CI, 0.42–0.93), even though no association was detected between high IgE (>100 kU/L) and glioma (OR = 0.98, 95% CI, 0.61–1.56) [30]. A prospective case-control study with a nested design including a higher number of cases (n = 275) has also demonstrated that the risk of glioma is inversely correlated to IgE response to inhalant allergens (OR = 0.73, 95% CI, 0.51–1.06) [31]. This relationship is particularly pronounced in women (OR = 0.53, 95% CI, 0.30–0.95) and the lowest OR was found in samples with the highest serum IgE levels [31]. Last, a nested case-control study with serum samples from 594 glioma and 374 GBM cases has shown that high levels of total IgE are associated with a significantly reduced risk of glioma, while allergen-specific IgE levels are correlated with a decreased risk of GBM specifically in women, but not in men [32]. Of note, this inverse association is present at least 20 years before tumor diagnosis [32].

A few studies have addressed the interplay between allergic inflammation and gliomagenesis with strategies different from the epidemiological approach. First experimental evidence can be found in initial attempts of immunotherapy. In a mouse model of glioma elicited in nude mice by the injection of U87 human glioma cells, the co-transplantation of an IL-4-secreting cell line promoted a significantly increased survival and a massive infiltration by eosinophils [33]. In line with these data, a phase I/II

clinical trial has shown that the intracavitary injection of IL-2 and lymphokine-activated killer (LAK) cells in GBM patients leads to improved survival, associated with eosinophilic infiltration of the central nervous system (CNS) [34]. Several gene therapy approaches by our group and others have shown that IL-4 delivery in immunocompetent hosts with malignant gliomas results in a significant impairment of tumor growth and prolonged survival [35–37]. In particular, we found that local production of IL-4 at the tumor site is associated with increased infiltration of CD8+ T and CD4+ T cells, B lymphocytes and macrophages [37]. Later, we have shown that transplantation of neural stem/progenitor cells retrovirally engineered to produce IL-4 in C57BL/6 mice with established GBM leads to the survival of most tumor-bearing mice [38]. These data, obtained in both experimental models and human disease, suggest that the induction of an IL-4 and/or eosinophil-mediated immune response might be beneficial to counteract tumor development.

In addition, mast cells (MCs) have been implicated in the pathogenesis of glioma. MCs constitutively express the high affinity receptor for IgE (FcεRI) and represent the key effector players in allergic reactions, when the re-exposure to a previously encountered antigen induces IgE-mediated anaphylactic degranulation and the consequent release of massive amounts of pre-formed mediators, such as histamine, cytokines (e.g., tumor necrosis factor (TNF), IL-4), chemokines, leukotrienes and growth factors (e.g., vascular endothelial growth factor, VEGF) [39]. Furthermore, monomeric IgE in the absence of antigen, can be even more effective than IgE plus antigen to induce MC-mediated release of inflammatory cytokines such as IL-4, IL-6 and TNF [40]. Together with microglia, MCs represent a key immune subset resident within the CNS, where they are mainly localized in the thalamus, hypothalamus and leptomeninges [41]. Significantly increased numbers of MCs have been detected in GBM tissue specimens in comparison to low-grade glioma (grade II), displaying often a perivascular localization and staining for CXCL12 and CXCR4 [42]. Similar findings have been obtained in an immunocompetent mouse model of glioma, elicited with the retroviral system RCAS/TV-a of gene delivery. In detail, the RCAS retrovirus encoding for two oncogenes (*KRas* and *Akt*) has been administered postnatally in *Ntv-a* and *Gtv-a* transgenic mice, which express the TV-a receptor for RCAS retrovirus under the control of Nestin or GFAP promoters, respectively, to obtain retroviral infection (and consequent oncogene expression) specifically in neural/glial progenitor cells or astrocytes. These *Ntv-a* and *Gtv-a* mouse lines carried at the same time deficiencies in tumor suppressor genes *Ink4a* or *Arf*, which are often deleted in human gliomas and developed gliomas of various grades and types [42]. The mouse strain with high-grade glioma (*Arf* −/−) displayed a significantly higher infiltration of MCs than *Ink4a*−/− mice developing low-grade gliomas and exhibited also a strong expression of stem cell factor (SCF), the main growth factor for MCs, around tumor blood vessels [42]. Interestingly, primary human gliomas have been shown to express SCF in a grade-dependent manner [43]. The same research group has later extended these findings to a larger cohort of patients (n = 188), confirming an accumulation of MCs in high-grade (i.e., grade III and IV) vs. low-grade (grade II) gliomas [44]. By taking advantage of a transwell migration assay, human glioma cell lines were shown to potently attract MCs by a mechanism partially dependent on the chemoattractant macrophage migration inhibitory factor (MIF), plasminogen activator inhibitor 1 (PAI-1) and the phosphorylation of STAT5 [44,45]. Of note, a significantly positive correlation was observed between the number of MCs and the cytoplasmic intensity of MIF staining in glioma tissue samples [44].

Notably, a recent analysis from lower grade gliomas showed that allergy is a robust, prognostic factor independent from other major prognostic molecular markers [46].

#### **4. Conclusions and Future Perspectives**

Type 2 immunity is recently emerging as a specific arm of the immune system physiologically deputed to defense against toxic and carcinogenic agents [5]. Data so far have suggested that at least some components of allergic inflammation might counteract the development of gliomas and GBM (Figure 1).

**Figure 1.** Schematic representation of the components of allergic inflammation with potentially anti-glioma activity. The establishment of a Th2-driven immune response against the tumor might support resistance to glioma growth through several modalities. The overexpression of the prototypical Th2-cytokine interleukin (IL)-4 within tumor microenvironment has been shown to counteract gliomagenesis [33,35–38,47]. This anti-tumor effect is associated with enhanced infiltration of immune cells such as CD8+ T lymphocytes, CD4+ T cells, B cells and monocytes at the tumor site (1) [37]. Th2 cells also secrete IL-5, a key cytokine for the recruitment and activation of eosinophils. Eosinophilic infiltrates within glioma tissue have been detected in early attempts of immunotherapy displaying a positive outcome [33,34] (2). IL-4 and IL-13 derived from Th2 cells can promote IgE isotype class switch recombination in B cells and their maturation to plasma cells. Theoretically, antigen-specific IgE might promote direct tumor killing through ADCC (3), similar to what was observed in a mouse model of ovarian cancer [48]. Alternatively, the engagement of FcεRI on mast cells (MCs) by monomeric IgE is known to induce the release of selective inflammatory cytokines [40], such as IL-4 and tumor necrosis factor (TNF), both endowed with anti-glioma activity [38,49] (4).

Epidemiological investigations have provided a huge amount of data showing an inverse association between allergy and gliomas, even though they do not provide any information on the causative relationship between these two conditions. In principle, it is possible that the tumor itself promotes immune-suppression of type 1 immunity and indirectly favors an imbalance of systemic immunity toward a Th2/allergic profile. However, the timing of most epidemiological observations, anticipating the clinical emergence of gliomas of many years, is in some contradiction with this hypothesis, at least in cases of primary GBM that are known to be formed in months rather than years. Moreover, some genetic variants in *IL13* and *IL4RA* genes confer opposite odds ratios for asthma and GBM development [27]. Given that germline polymorphisms were utilized as biomarkers of susceptibility to asthma in this study, these results cannot be ascribed to an effect of GBM on the immune system [27]. According to the cancer immunoediting hypothesis, the interaction between the immune system and tumor cells undergoes three different stages termed respectively "elimination", "equilibrium" and "escape" [50]. The elimination phase is characterized by the interplay of innate and adaptive immune mechanisms, which drives the destruction of tumor cells from the host. However,

if this process is not complete, rare cancer cell variants might enter an equilibrium stage, in which tumor growth is still prevented by adaptive immunity and the tumor is subject to constant editing of its immunogenicity. During this stage, immune pressure on genetically-unstable tumor cells might result in the selection of tumor variants endowed with the ability to escape immune surveillance and form clinically visible tumors [50]. In the context of brain tumors, it's possible to speculate that the genetic background favoring the development of allergic inflammation might support the elimination phase and reduce the probability of developing glioma over the course of time.

Immunotherapeutic approaches have shown that IL-4 can counteract glioma growth and eosinophilic infiltration of tumor tissue correlates with a positive outcome of immunotherapy. Nonetheless, the mechanisms through which these typical components of allergic inflammation might exert an anti-tumoral effect are not clearly understood.

Higher levels of IgE are associated with a reduced risk of glioma. IgE is endowed with tumorkilling properties by mediating antibody-dependent cell-mediated cytotoxicity. Its effect has been shown in a mouse model of ovarian cancer [48], but no study has investigated whether IgE is reactive against tumor antigens in glioma patients. Furthermore, IgE alone or in combination with an antigen can stimulate MC functions.

Studies describing the accumulation of MCs in GBM have offered important evidence of the potential involvement of these crucial effector cells of allergy in gliomagenesis, however, whether MCs promote or counteract tumor growth is not known and should be subject to further investigation. According to the modality of activation, MCs have been shown to promote or break immune tolerance. This concept is illustrated in models of allotransplantation and CD8+ T cell-mediated graft rejection. Indeed, IL-9 secreted by regulatory T (Treg) cells has been shown to recruit and activate MCs to promote regional immune suppression and acceptance of allograft skin transplants [51]. Conversely, in the same model when MCs undergo anaphylactic degranulation, they break Treg-mediated suppression and induce allograft rejection [52]. To understand the contribution of MCs to gliomagenesis, it might be worthwhile to evaluate the development of gliomas in mouse models of MC-deficiency [53]. Interestingly, recent work has shown that *Hdc*−/− mice, that genetically lack histamine, exhibit increased glioma growth and reduced survival, associated with the accumulation of immunosuppressive immature myeloid cells [54]. Of note, MC granules represent the main immunological source of histamine and *Hdc*−/− mice display several deficits in MC numbers, morphology and granular contents [55]. In addition, there are data supporting the contribution of MCs to tumor formation in the context of neurofibromatosis type 1 [56].

**Author Contributions:** Both authors contributed in the conceptualization, writing and final revision of the manuscript.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

© 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

## **Combined Amino Acid Positron Emission Tomography and Advanced Magnetic Resonance Imaging in Glioma Patients**

**Philipp Lohmann 1,\* , Jan-Michael Werner 2 , N. Jon Shah 1,3,4, Gereon R. Fink 1,2, Karl-Josef Langen 1,5 and Norbert Galldiks 1,2,6**

Received: 4 January 2019; Accepted: 25 January 2019; Published: 29 January 2019

**Abstract:** Imaging techniques such as positron emission tomography (PET) and magnetic resonance imaging (MRI) provide valuable information about brain tumor patients. Particularly amino acid PET, advanced MRI techniques, and combinations thereof are of great interest for the non-invasive assessment of biological characteristics in patients with primary or secondary brain cancer. A methodological innovation that potentially advances research in patients with brain tumors is the increasing availability of hybrid PET/MRI systems, which enables the simultaneous acquisition of both imaging modalities. Furthermore, the advent of ultra-high field MRI scanners operating at magnetic field strengths of 7 T or more will allow further development of metabolic MR imaging at higher resolution. This review focuses on the combination of amino acid PET with MR spectroscopic imaging, perfusion- and diffusion-weighted imaging, as well as chemical exchange saturation transfer in patients with high-grade gliomas, especially glioblastomas.

 Introduction**

At present, contrast-enhanced magnetic resonance imaging (MRI) is the method of choice for brain tumor diagnostics since MRI provides excellent soft tissue contrast, comparatively high resolution, and is widely available. On the downside, its specificity for neoplastic tissue is low, hampering the evaluation of tumor extent in both enhancing and non-enhancing gliomas as well as the differentiation of tumor progression from non-specific treatment-related changes [1–3].

[ 18F]-2-Fluoro-2-deoxy-D-glucose (FDG) is the most extensively used positron emission tomography (PET) tracer to date and has hence gained exceptional importance in general oncology. In stark contrast, its applicability in brain tumor diagnostics is hindered by high levels of physiological glucose uptake in the cerebral cortex resulting in diminished contrast between tumor and background. On the contrary, the cerebral uptake of radiolabeled amino acids is low while it is typically increased

in brain tumors, resulting in an enhanced tumor-to-background contrast [4,5]. Importantly, the uptake of amino acid tracers is independent of the blood-brain barrier integrity, allowing the evaluation of amino acid uptake in non-enhancing gliomas [1,6]. Additionally, amino acid PET has demonstrated its usefulness for the differentiation of tumor progression from treatment-related changes [7–12] and for various other indications, e.g., treatment monitoring, prognostication or tumor grading [4–6,13–17]. Among amino acid tracers labelled with carbon-11 used for PET imaging in neuro-oncology, [ 11C]-methyl-L-methionine (MET) is currently best evaluated [18,19]. However, the short half-life of carbon-11 (20 min) limits its use to centers with an on-site cyclotron. To overcome such logistical limitations, amino acid tracers have been labeled with fluorine-18, which has a longer half-life of 110 min [4,6]. For that reason, O-(2-[18F]-fluoroethyl)-L-tyrosine (FET) has replaced MET in many neuro-oncological centers. Subsequently, FET has become the most widely used radiotracer for brain tumor diagnostics, especially in Western Europe. Another fluorine-18 labeled amino acid tracer is 3,4-dihydroxy-6-[18F]-fluoro-L-phenylalanine (FDOPA), originally developed to evaluate the dopamine synthesis in the basal ganglia, which also shows increased uptake in brain tumors [20,21]. Although brain tumors distant to the striatum are depicted with high contrast, the high striatal uptake of FDOPA limits its usefulness for tumor delineation especially near the basal ganglia [22,23]. The Response Assessment in Neuro-Oncology (RANO) working group considers the additional value of amino acid PET for brain tumor diagnostics as highly relevant and recommends its use at every stage of brain tumor management [24].

The combination of MRI and PET, especially if hybrid PET/MR scanners are used, offers great potential for brain tumor diagnostics. This technology is particularly attractive when ultra-high field MRI scanners operating at magnetic field strengths of 7 T or more are widely available, allowing comparative anatomical and metabolic imaging at high resolution. Currently, amino acid PET tracers are the best evaluated and most frequently used PET tracers for brain tumor diagnostics. Therefore, this review focuses on the combination of amino acid PET with MR spectroscopy (MRS), perfusionand diffusion-weighted imaging (PWI, DWI), and chemical exchange saturation transfer (CEST) in patients with glioblastoma (GBM).

## **2. Search Strategy**

A PubMed search of the published literature with the combination of the search terms "glioblastoma", "brain tumors", "high-grade glioma", "positron emission tomography", "magnetic resonance imaging", "magnetic resonance spectroscopy", "perfusion-weighted imaging", "diffusion-weighted imaging", "chemical exchange saturation transfer", "PET", "amino acid PET", "MRI", "advanced MRI", "MRS", "PWI", "DWI", "CEST", and "hybrid PET/MR" before and inclusive of December 2018 was performed. Additional literature was retrieved from the reference lists of all identified articles. Furthermore, articles identified through searches of the authors' files were included. Only papers in English were considered.

## **3. Amino Acid PET and MRS**

MR spectroscopy (MRS) is a non-invasive method to detect selected water-soluble metabolites in vivo. In magnetic resonance, an externally applied magnetic field experienced by the nucleus is modulated by the distribution of surrounding electrons. Given that every molecule presents with a slightly different distribution of electrons, every nucleus experiences a slightly different magnetic field.

Consequently, every molecule has its characteristic magnetic field 'signature' that, combined with interactions amongst the nuclei, results in slightly different resonance frequencies leading to differential signals. These signal differences are used in MRS to identify the metabolites of interest. Every nucleus possessing a non-zero spin can be theoretically used for MRS, e.g., protons, carbon-13 and phosphorous-31. However, due to the low abundance of the latter and limited resolution of metabolite profiles in vivo, mainly proton spectra are used in clinical practice.

Metabolites that are frequently assessed by MRS are lactate, lipids, alanine, N-acetylaspartate (NAA), glutamine, glutamate, 2-hydroxyglutarate (2-HG), citrate, creatine, choline, and myo-inositol. In gliomas, with increasing grade of malignancy, NAA and creatine are usually decreased, whereas choline, lipids and lactate are increased [25–27]. Furthermore, the accumulation of 2-HG caused by gene mutations encoding for the enzyme isocitrate dehydrogenase (IDH) can be detected by 2-HG spectroscopy at 3 T [28]. Although several studies demonstrated the feasibility of 2-HG spectroscopy in a clinical setting [29–32], 2-HG spectroscopy remains challenging due to a small and complex signal and it is therefore not yet established in clinical routine [33].

Two different MRS methods exist using either data from a single voxel or multiple voxels in a single slice or multiple slices of the investigated organ. Single-voxel spectroscopy is the most widely used method due to simple data acquisition and relatively short scanning time. Moreover, the comparatively high signal-to-noise ratio achieved by single-voxel spectroscopy results in high-quality spectra enabling a quantitative analysis and straightforward interpretation. However, the manually preselected region of interest (ROI) based on information from T2-weighted or contrast-enhanced MRI may represent only a fraction of the tumor, potentially resulting in an incomplete evaluation of tumor biology.

Multi-voxel spectroscopy techniques may overcome these limitations by covering a more extended two- or three-dimensional ROI at higher spatial resolution. Especially in heterogenous lesions, this allows detection of subtle intratumoral signal alterations [34]. Another advantage of smaller voxels used in multi-voxel spectroscopy is the reduction of partial volume effects as structures such as cerebrospinal fluid or fat may diminish the quality of spectra and can be excluded from ROI definition. However, because of the difficulty of achieving good enough shimming over a large enough region, multi-voxel spectroscopy can have a lower signal-to-noise ratio, a reduced spectral quality for individual voxels, requires longer scanning times, and is technically more demanding than single-voxel spectroscopy [35].

In a few studies, MRS combined with amino acid PET in glioma patients was used. D´Souza and colleagues [36] evaluated in 29 high-grade glioma patients the diagnostic performance of MET PET and single-voxel MRS at 3 T for the correct diagnosis of tumor recurrence after neurooncological treatment including radiotherapy and chemotherapy. Metrics derived from both MET PET (i.e., tumor/brain ratios) and single-voxel MRS (i.e., choline/creatine ratios) suggested a high diagnostic accuracy of 85–90% for the detection of high-grade glioma recurrence. Floeth and co-workers [37] explored the use of combined FET PET and single-voxel MRS at 1.5 T in 50 patients with newly diagnosed lesions suspicious for gliomas on contrast-enhanced MRI. Based on the neuropathological assessment, gliomas could be identified with an accuracy of 68% using conventional MRI alone. Accuracy could be increased to 97% when conventional MR imaging was used in combination with FET PET and single-voxel MRS. In that study, FET lesion/brain ratios and NAA/choline ratios were identified as significant independent predictors for the identification of glioma tissue.

Stadlbauer et al. [38] used two-dimensional multi-voxel spectroscopy at 1.5 T in combination with FET PET to spatially correlate concentrations of choline, creatine, and NAA with FET uptake in 15 glioma patients. The authors found significant correlations between increased FET uptake and parameters derived from MRS. More precisely, increased FET uptake was significantly correlated with the extent of neuronal loss (NAA) and partially with membrane proliferation (choline), indicating that both PET and MRS provide complementary information. Yet, the number of patients included in that study was relatively small and two-dimensional MRS allowed only limited coverage of the tumor, especially in comparison with amino acid PET, which provided three-dimensional information concerning tracer distribution.

To overcome these limitations, Mauler and colleagues [39] investigated the spatial correlation between FET PET and three-dimensional MRS covering the whole brain. Forty-one glioma patients were investigated usinga3T hybrid PET/MR scanner. Importantly, the authors reported that the FET uptake and increased choline/NAA ratios were not consistently spatially congruent (Figure 1). Unfortunately, however, no spatial correlation with neuropathological information obtained via stereotactic biopsy was performed to further explore the differences between amino acid uptake and the metabolites detected by MRS in gliomas.

**Figure 1.** Simultaneously acquired contrast-enhanced (CE) T1-weighted MRI, FET PET and whole-brain MRS shows elevated choline/N-acetylaspartate (Cho/NAA) ratios in a patient with neuropathologically confirmed glioblastoma. Areas of contrast enhancement, FET uptake and increased Cho/NAA are spatially incongruent. Image courtesy of Joerg Mauler, Institute of Neuroscience and Medicine, Forschungszentrum Juelich, Germany, and Andrew Maudsley, Department of Radiology, University of Miami Medical School, Miami, FL, USA.

## **4. Amino Acid PET and PWI**

PWI is a non-invasive MRI technique to measure blood flow quantitatively. In Neuro-Oncology, the parameters relative cerebral blood volume (rCBV) and cerebral blood flow (rCBF) are frequently assessed. Most commonly, a gadolinium-based contrast agent is used to assess tissue perfusion. After i.v. injection, the passage of the contrast agent leads to i) a local magnetic field distortion (susceptibility effect) in the vicinity of the vessels causing a signal drop in T2- or T2\*-weighted MRI, also called dynamic susceptibility contrast (DSC), or ii) a shortening of T1-relaxation time causing a signal increase in T1-weighted MRI, also called dynamic contrast-enhanced (DCE) MRI. While DSC data requires only the first pass of intravascular contrast agent for assessing tissue perfusion, DCE additionally evaluates information about the continuous accumulation of the contrast agent in the extracellular space. Consequently, the acquisition times for DCE are longer than for DSC.

Another PWI method called arterial spin labelling (ASL) does not require a contrast agent. Here, endogenous water molecules in blood vessels are magnetically labeled by applying a specific radiofrequency pulse. Passage of these labeled molecules through the tissue of interest leads to a reduction of signal intensity in proportion to the perfusion. However, by dispensing with the administration of a contrast agent, the signal-to-noise ratio of ASL is inherently low, so that repetitive signal averaging is mandatory resulting in prolonged acquisition times. ASL significantly benefits from higher magnetic field strengths. Thus, ultra-high field MRI scanners operating at magnetic field strengths of up to 7 T may boost its clinical utility in the future.

Several studies combined PWI and amino acid PET in patients with high-grade gliomas for the delineation of the glioma extent. Filss and colleagues [40] compared DSC PWI and FET PET acquired concurrently using a 3 T hybrid PET/MR scanner in 56 glioma patients (24 patients with GBM). One main finding was that FET PET tumor volumes were significantly larger than tumor volumes delineated by rCBV maps. Furthermore, the spatial congruence of the two methods was poor, and the localization of tumor hotspots identified by PWI and FET PET yielded inconsistent results indicating that the delineation of glioma extent is not appropriately reflected by PWI and hence cannot replace amino acid PET (Figure 2). These findings were confirmed subsequently [41,42]. Besides, similar

results were reported by Cicone and co-workers for the comparison of DSC PWI at 1.5 T and FDOPA PET [22].

**Figure 2.** Simultaneously acquired contrast-enhanced (CE) T1-weighted MRI, FET PET and relative cerebral blood volume (rCBV) obtained from PWI in a patient with neuropathologically confirmed glioblastoma. FET PET identifies metabolically active tumor areas without an increased rCBV. Image courtesy of Christian Filss, Institute of Neuroscience and Medicine, Forschungszentrum Juelich, Juelich, Germany.

Verger et al. [43] investigated the usefulness of FET PET and DSC PWI at 3 T for the grading of gliomas in 72 patients with newly diagnosed glioma. The diagnostic accuracy for glioma grading was comparable for both FET PET and rCBV with an area under the receiver-operating characteristic curve of about 0.80. However, neuropathological diagnoses were not based on the revised WHO classification from 2016 [44], which limits the applicability of the results. The authors also found in 78% of patients a considerable spatial disparity between the local hotspots delineated using the two methods, consistent with previous studies [22,40,41].

Dandois and co-workers [45] compared the diagnostic accuracy of DSC PWI at 1.5 T and amino acid PET using MET for the differentiation of recurrent glioma from radiation necrosis in 28 high-grade glioma patients. The diagnostic performance was comparable between rCBV maps and MET PET for this clinically relevant question. These findings were reinforced by other studies [36,46,47]. However, in the majority of the cases, the diagnosis of either recurrent glioma or radiation necrosis was based on clinical observation and radiological follow-up rather than neuropathology. In stark contrast, Verger and colleagues [48] reported that FET PET seems to be superior to PWI at 3 T to diagnose recurrent glioma. Importantly, and in contrast to the latter studies mentioned above, the definite diagnosis was based on neuropathology in about 80% of the cases. These findings were confirmed by another study [49]. Subsequently, Roodakker et al. [50] compared regional MET PET and PWI at 3 T with local neuropathology in patients with en bloc-resected oligodendrogliomas. They showed that MET uptake correlated with tumor cell density throughout the entire tumor volume in all patients. Interestingly, tumor perfusion (rCBV) did not correlate with MET uptake or with any other histological marker.

Morana and colleagues [51] compared FDOPA PET and ASL at 1.5 T for the grading and prediction of tumor progression in 26 pediatric gliomas. Authors reported a better diagnostic performance for FDOPA PET with an area under the receiver-operating characteristic curve of 0.95 compared to 0.88 for rCBF derived from ASL.

The reported differences across the studies highlight the need for prospective studies with larger cohorts and neuropathological validation to further evaluate the differences between perfusion MRI and amino acid PET. Furthermore, a more recent meta-analysis regarding PWI in high-grade gliomas by Patel and co-workers [52] revealed a considerable heterogeneity of applied MR sequences, acquisition and post-processing parameters utilized in PWI studies of brain tumors thereby hampering comparability and reproducibility of results.

## **5. Amino Acid PET and DWI**

Diffusion-weighted imaging (DWI) is an MRI technique based on the measurement of Brownian motion of water molecules to generate image contrast. DWI contrast uses two opposing gradient pulses; the first one induces a phase shift in water molecules, leading to a signal reduction. Subsequently, a second opposed gradient pulse is applied, which rephases the water molecules in the region of interest, leading to a recovery of the water signal. If water molecules moved out of the region of interest within the time between the two pulses, the number of water molecules contributing to the water signal is reduced. Therefore, the signal intensity is decreased. By variation of the amplitude, pulse duration, and time between the pulses, the degree of diffusion-weighting, represented by the b-value, can be altered. The apparent diffusion coefficient (ADC) is calculated from different b-values (typical range of b-values, 0–1000 s/mm2), and represents a quantitative measure of diffusivity [53]. In brain tumors, hypointense signals in ADC maps (indicating lower ADC values) are suspicious for neoplastic tissue due to high tumor cellularity leading to a restriction of diffusivity [6].

Several studies addressed the spatial congruency of DWI and amino acid PET in glioma patients with mixed results. Karavaeva and colleagues [54] demonstrated a significant spatial relationship of FDOPA uptake and ADC signal alterations at 1.5 T in patients with recurrent high-grade glioma. Furthermore, a significant spatial correlation of both ADC values and FDOPA uptake with the cellular proliferation rate was reported. However, it remains unclear whether the cellular proliferation rate was associated with tumor progression or treatment-related tissue reactions such as inflammatory processes. On the other hand, several other studies reported contradictory results concerning the spatial congruency of amino acid uptake and ADC abnormalities [55–57]. Recently, Popp and co-workers [57] showed that tumor volumes in patients with recurrent glioblastoma delineated using ADC maps acquired at 1.5 T were frequently localized outside of areas with increased FET uptake. Another study by Kinoshita [58] and colleagues reported that MET PET demonstrated a more robust and reliable estimation of cell density in gliomas compared to ADC maps acquired at 3 T. These controversial study results concerning the spatial congruency are most likely related to the fact that amino acid PET and DWI encode different biological properties [57,59] (Figure 3).

**Figure 3.** Contrast-enhanced (CE) T1-weighted MRI, FET PET and the apparent diffusion coefficient (ADC) map obtained from DWI in a patient with neuropathologically confirmed glioblastoma. In contrast to the ADC map, FET PET shows the metabolically active tumor tissue with high contrast. Furthermore, the ADC map shows slightly increased diffusivity in various subregions, erroneously indicating treatment-related changes.

Pyka and colleagues [49] compared FET PET and DWI at 3 T for the differentiation of tumor progression from treatment-related changes in 47 high-grade glioma patients. The authors reported a superior performance for FET PET compared to ADC (AUC, 0.86 vs. 0.69). The combination of FET PET and ADC with other MRI metrics such as rCBV resulted in an AUC improvement to 0.89.

It should be pointed out that DWI has a low spatial resolution and a poor signal-to-noise ratio. Although stronger gradients and higher magnetic field strengths can increase signal-to-noise ratios, susceptibility artifacts would become concurrently more pronounced. Another limitation of DWI is the compromised reproducibility and comparability of ADC values, even when the same MR scanner is used [60]. Moreover, diffusivity can also be altered by ischemia, infection, inflammation, gliosis, necrosis, or vascular proliferation, suggesting non-specificity of DWI changes [6,61,62].

#### **6. Amino Acid PET and CEST**

Chemical exchange saturation transfer (CEST) is an MRI technique for the indirect detection of different metabolites using the MR free water signal [63]. CEST contrasts are based on the spontaneous exchange of free water protons and protons bound to metabolites (solute-bound protons). After saturation of solute-bound protons by the application of a radio-frequency pulse with a specific frequency depending on the metabolite of interest, these protons exchange with free water protons and cause a signal reduction of free water. Thus, the CEST signal depends upon the exchange rate between free water and solute-bound protons and the concentration of the metabolite of interest [64,65].

CEST has already been applied for the measurement of glycogen (GlycoCEST) [66], glucose (GlucoCEST) [67], glutamate (GluCEST) [68], amide proton transfer (APT) [69], and intracellular pH [69–71]. The signal used to generate CEST contrasts is complex and small. Therefore, CEST imaging may benefit from higher magnetic field strengths (> 3 T) due to an increased signal-to-noise ratio and spectral dispersion. However, complex data processing steps are required to evaluate metabolite concentrations from CEST contrasts [72–75].

Although several studies evaluated CEST in patients with GBM [75–77], only few studies directly compared amino acid PET and CEST in more than 5 patients. Da Silva et al. [78] compared FET PET and APT CEST MRI in 8 high-grade gliomas patients using a 3 T hybrid PET/MR scanner. The authors showed that FET PET and APT CEST are spatially incongruent and reflect different biological information. However, although CEST and amino acid PET were acquired simultaneously, the number of patients was low, and no correlation with local neuropathology was obtained.

#### **7. Conclusions and Outlook**

The combination of amino acid PET and advanced MRI methods offers a variety of new vistas for the assessment of brain tumors with the potential to overcome the limitations of conventional MRI. Amino acid PET and advanced MRI methods already provided promising results for the clinical management of patients with GBM. Both the complementary and additive value of these methods as well as their incongruent findings, suggesting differential biological information, warrant further investigation including neuropathological validation. The increasing advent of hybrid PET/MR scanners offers the great potential of comparative studies using both amino acid PET and advanced MRI in a single session. Furthermore, the clinical benefits of simultaneous PET/MR imaging need to be balanced against the relatively high cost of such an approach. Of note, the increasing availability of ultra-high field MRI scanners with magnetic field strengths of 7 T will help to develop novel techniques thereby promoting advanced MRI in neuro-oncology as almost all contrasts benefit from the higher spatial resolution related to the increased signal-to-noise ratio.

Amino acid PET is a robust and attractive approach for clinicians for many reasons including easy scan reading. Furthermore, most studies using amino acid PET in neuro-oncology provide comparable results across different scanners, which is also a consequence of national and international efforts concerning the standardization of amino acid PET acquisition and evaluation in brain tumor imaging [24,79]. Recently, joint practice guidelines were developed by the European Association of Nuclear Medicine (EANM), the Society of Nuclear Medicine and Molecular Imaging (SNMMI), the European Association of Neuro-Oncology (EANO), and the working group for Response Assessment in Neuro-Oncology with PET (PET-RANO) [80].

In contrast, the yet missing standardization of advanced MRI methods is—at least in part—likely to account for conflicting data. In a significant number of advanced MRI studies, self-developed or self-optimized MR sequence protocols and acquisition parameters, as well as post-processing tools, were used, thereby impeding comparability and reproducibility of the results. Besides, vendors of MRI and PET scanners should strive for further standardization of imaging protocols and data processing since better comparability and easier reproducibility of the data is likely to boost their clinical value. Finally, further implementation of various advanced MRI methods as well as amino acid PET in clinical routine requires the validation of neuroimaging findings through neuropathology.

To conclude, advanced MRI in combination with amino acid PET has the potential to become an essential diagnostic tool for improving the clinical management of patients with high-grade gliomas, as well as in light of emerging high-throughput analysis methods of large-scale data sets such as radiomics and machine learning [16,81,82].

**Funding:** This research received no external funding.

**Conflicts of Interest:** The authors declare no conflict of interest.

## **References**

© 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

## **Treatment of Glioblastoma (GBM) with the Addition of Tumor-Treating Fields (TTF): A Review**

## **Denise Fabian 1, Maria del Pilar Guillermo Prieto Eibl 2, Iyad Alnahhas 2, Nikhil Sebastian 1, Pierre Giglio 2, Vinay Puduvalli 2, Javier Gonzalez 2 and Joshua D. Palmer 2,\***

Received: 14 January 2019; Accepted: 31 January 2019; Published: 2 February 2019

**Abstract:** Glioblastoma (GBM) is the most common primary brain tumor. Despite aggressive treatment, GBM almost always recurs. The current standard-of-care for treatment of newly diagnosed GBM has remained relatively unchanged since 2005: maximal safe resection followed by concomitant chemoradiation (CRT) with temozolomide (TMZ), and subsequent adjuvant TMZ. In 2011, the first-generation tumor treating fields (TTF) device, known at the time as the NovoTTF-100A System (renamed Optune), was approved by the Food and Drug Administration (FDA) for treatment of recurrent GBM. The TTF device was subsequently approved as an adjuvant therapy for newly-diagnosed GBM in 2015. The following is a review of the TTF device, including evidence supporting its use and limitations.

**Keywords:** glioblastoma; GBM; tumor treating fields; TTF

## **1. Background**

Glioblastoma (GBM) is the most aggressive and common primary brain tumor. Treatment remains challenging, as GBM inevitably recurs despite surgical debulking, radiation and chemotherapy. The current standard-of-care is comprised of maximal safe resection—gross total resection if feasible—followed by chemoradiation (CRT). Radiation therapy consists of 60 Gray (Gy) in 30 fractions over a period of 6 weeks with concomitant daily temozolomide (TMZ), followed by adjuvant TMZ (days 1–5 every 28 days). Median survival ranges from twelve to fifteen months following diagnosis and treatment [1]. The current five-year survival rate is about five percent in the United States [2].

Since 2005, several clinical trials have been conducted attempting to improve outcomes for GBM patients. For example, the Radiation Therapy Oncology Group (RTOG, Philadelphia, PA, USA) 0525 was a phase III trial that compared conventional adjuvant TMZ with dose-dense (dd) TMZ. Despite confirming the prognostic significance of *MGMT* promotor methylation, survival did not improve with dd TMZ [3]. The addition of Bevacizumab in RTOG 0825 demonstrated improvement in progression free survival (PFS), however, it did not yield changes on overall survival (OS) [4,5]. The addition of Everolimus, an oral mammialian target of rapamycin (mTOR) inhibitor, to chemoradiation, increased treatment-related toxicities, and did not have any impact on progression-free survival (PFS) and it even shortened the OS [4]. Additional trials are being conducted to look into the use of checkpoint inhibitors, such as Ipilimumab and Nivolumab (NRG-BN002) and radiation dose-escalation with photon Intensity modulated radiotherapy (IMRT) or Proton Beam Therapy (NRG-BN001) NRG Oncology is a National Clinical Trials Network group created through the efforts of the National Surgical Adjuvant Breast and Bowel Project (NSABP), the RTOG, and the Gynecologic Oncology Group (GOG). Recently, a Phase

II Trial of Neoadjuvant TMZ followed by accelerated hypofractionated radiation therapy (60 Gy in 20 fractions) demonstrated a median OS of twenty-two months with a PFS of 13.2 months, comparing favorably to OS previously reported in other clinical trials [6].

In 2011, the United States Food and Drug Administration (FDA) approved a tumor treating fields (TTF) device for treatment of recurrent or refractory GBM. More recently, the FDA approved the TTF device as adjuvant treatment for newly-diagnosed patients after completing standard-of-care surgery and chemoradiation. The National Comprehensive Cancer Network (NCCN) added the TTF device as an option for treatment of newly-diagnosed GBM. Despite FDA approval, skepticism remains regarding this therapy. In this review we discuss the current evidence supporting treatment with the TTF device and its limitations.

## **2. Materials and Methods**

We conducted a comprehensive literary investigation utilizing PubMed and Google search engines. Approximately 50 journal articles, newspaper articles, and abstracts were reviewed. Ultimately, 43 sources were selected for relevance and impact. Relevance of topics was selected based on talking points at the 2018 American Society for Radiation Oncology (ASTRO) conference and common questions proposed by the patients and practicing clinicians at our institution.

## *2.1. Tumor Treating Fields Device Proposed Mechanism*

The TTF device includes four transducer arrays, each consisting of nine insulated electrodes which are applied to the patient's scalp to deliver low-intensity, intermediate-frequency (100–300 kHz) alternating electric fields [7,8]. In 2004, a preclinical model demonstrated the inhibitory effect of the TTF device on proliferating cells whereas nonproliferating cells remained unaffected. Treatment with the TTF device is thought to interfere with normal polymerization and depolymerization of microtubules of the mitotic spindle by positioning tubulin dimers further away from the growing end of the microtubules [8]. This leads to mitotic disruption, which leads to mitotic catastrophe and ultimately to mitotic cell death. The investigators demonstrated this by setting up melanoma cell cultures in vitro with TTFs generated by pairs of insulated wires. In cells exposed to TTF, significant inhibition of growth was seen after 24 h exposure. This effect was also seen beyond the exposure time [8]. To explore the effects of TTF on molecular processes, the investigators used time-lapse microphotography. In cells treated with TTF, mitosis began normally, but was prolonged [8]. Additionally, a quarter of cells in the TTF cultures were destroyed during the formation of the mitotic cleavage furrow [8]. Finally, nuclear rotation was seen in the TTF cultures [8]. The investigators explain that microtubules in dividing cells have electric dipole moments, which may be altered by the forces exerted by TTF [8]. They showed this by comparing the movement of cellular microtubules by fixing the cells after 24 h of TTF vs. no treatment; when the fixed cells were viewed under fluorescence microscopy, more than 50% of the TTF treated cells had abnormal mitosis compared to less than 5% of the control cells [8]. The investigators defined two mechanisms of action: (1) disruption of the polar tubulin molecule orientation, pushing tubulin dimers further than 14 nm away from the growing end of the microtubule and thereby interfering with proper microtubule assembly; and (2) cell destruction by pulling of all intracellular and polar particles towards the mitotic cleavage furrow, resulting in a pile-up that interferes with cytokinesis [8].

Further laboratory studies demonstrated similar cancer growth inhibition in multiple in vitro cell lines and animal tumor models. A single-arm pilot clinical trial was performed in ten patients shortly thereafter [9]. Novocure Ltd. (St. Helier, NJ, USA) manufactured a clinical TTF device, named Optune (formally Novo TTF-100A, St. Helierl, NJ, USA). It utilizes electrodes configured as a cap that is placed on the patient's shaved scalp and powered by a battery package [10]. At present, the proposed mechanism also includes interference with Septin fibers of proliferating cells as well as endoplasmic reticulum stress leading to cellular stress and autophagy [11,12]. This demonstrates that the effect of TTF on interactions of intercellular may be more complex than originally thought; similarly, the effects on many other intracellular proteins are unknown. Further studies are warranted in both cancer and normal cell lines.

## *2.2. Treatment of Glioblastoma with Tumor Treating Fields*

The first phase III clinical trial testing the TTF device (EF-11) was published in 2012 and included 237 patients with recurrent GBM [13]. This study compared the first generation of the TFF device alone—worn 18–24 h a day—to chemotherapy of the treating physician's choice. The median survival was 6.6 vs. 6.0 months (*p* = 0.27) for patients subject to treatment utilizing the TTF device or chemotherapy, respectively. Although the trial did not demonstrate its primary endpoint of improved overall survival, efficacy was similar to commonly used chemotherapy regimens. Given the localized nature of the treatment, a lower toxicity profile and better quality of life—including improved cognition and emotional well-being—were reported in the TTF arm. These patients also experienced less chemotherapy-related side effects, such as hematologic toxicity, gastrointestinal adverse events, and infections. Further analysis found that compliance with therapy was linked to outcomes; more specifically device compliance above 75% correlated with improved OS [14]. It is important to note that the trials were not powered to look into these subgroup analyses. The limitations of the study included absence of a placebo control arm and a heterogeneous patient population who had received various chemotherapy treatments prior to the trial. The results from this trial lead to the 2011 FDA approval of the first generation of the TFF device for treatment of patients with recurrent GBM or GBM that has not responded to traditional therapy [15]. Approval of this device was contingent on Novocure conducting additional studies and clinical trials to demonstrate device efficacy [16].

Following the trial, the Patient Registry Data set (PRiDe)—a registry of all patients with recurrent GBM who received therapy with the TTF device—was used to analyze the clinical outcomes of TTF device use in 91 cancer centers across the United States [17]. Compared to patients in the EF-11 trial, the 457 recurrent GBM patients in the PRiDe data set were more likely to use the TTF device for their first recurrence (33% vs. 9%). Median OS was improved in clinical practice when compared to the EF-11 trial (9.6 months vs. 6.6 months, hazard ratio (HR) 0.66, *p* = 0.003). As seen previously, device compliance rate of 75% or greater was associated with a significantly improved overall survival when compared to device compliance rate of less than 75% (7.7 vs. 4.5 months, *p* = 0.042). There were no significant adverse events, and the most common side effect was skin reaction [17].

In 2009, a multi-center phase III clinical trial lead by Dr. Roger Stupp was initiated for newly diagnosed GBM, studying the addition of TTF device treatment to maintenance TMZ (EF-14 trial) [7]. This study randomized 695 GBM patients in a 2:1 format to receive TTF device treatment plus maintenance TMZ or TMZ alone, following standard-of-care surgery and concurrent chemoradiotherapy. Patients with a Karnofsky performance score (KPS) of less than seventy, evidence of progressive disease following chemoradiotherapy, infratentorial tumor location or severe comorbidities were excluded from the study. An interim analysis presented in 2015 demonstrated improved PFS (7.1 months in the TTF device plus TMZ group and 4.0 in the TMZ alone group (*p* = 0.001) [7]. The final study was published in 2017, which reported an improved median overall survival of 20.9 months in the TTF device plus TMZ group vs. 16.0 months in the TMZ only group (*p* < 0.001). Of note, the reported survival times were measured from time of randomization, which was done after completion of chemoradiation, and which was about 3.8 months from original diagnosis. About half of patients who received treatment with the TFF device experienced mild to moderate skin toxicity [7]. Accordingly, the FDA approved the use of the TTF device for use in newly diagnosed GBM on 5 October 2015 [15]. Both Phase III clinical trials using TTF for GBM are summarized in Table 1.

This publication lead to the National Comprehensive Cancer Network (NCCN) adaptation of TTF treatment to the Clinical Practice Guidelines in Oncology for Central Nervous System. Additional analysis of the EF-14 trial has shown that the overall survival (OS) is improved at five years, regardless of other prognostic factors, such as age, performance status, extent of resection, and neurologic status. An abstract presented at ASTRO 2018 showed that that use of the TTF device and TMZ improved overall survival out to five years in all three recursive partitioning analysis (RPA) classes [18].

As expected, further clinical investigations using TFF are well underway (Table 2). In November 2018, a phase II trial opened comparing a combination treatment with the TTF and nivolumab with or without ipilimumab in patients with bevacizumab-naive recurrent GBM [19]. The trial is expected to close in 2021 and has an accrual goal of sixty (60) patients. Table 2 is a summary of ongoing trials using TTF in GBM. Research on utilization of TTF in cancer care is also not limited to GBM. A phase 2 pilot study on the safety and efficacy of the TTF device concomitant with pemetrexed and cisplatin or carboplatin in malignant pleural mesothelioma (STELLER) recently closed in April 2018. In this study, 150 kHz electrode arrays were placed on the thorax of patients with previously treated malignant pleural mesothelioma in addition to chemotherapy. The preliminary efficacy will be compared to historical cohorts [20]. TFF devices are being explored in disease sites such brain metastasis secondary to non-small cell lung carcinoma, ovarian carcinoma, pancreatic carcinoma, meningioma and even as an alternative to prophylactic cranial irradiation in small cell lung cancer [21,22].

**Number of**

1 Abbreviations: GBM: glioblastoma multiforme, CHT: chemotherapy, TMZ: temozolomide, TTF: tumor treating fields, OS: overall survival, PFS: progression-free

 survival.

**Table 2.** Ongoing trials using TTF for GBM as of December 2018 1.

Abbreviations: GBM: glioblastoma multiforme, TTF: tumor treating fields, ORR: objective response rate, OS: overall survival, PFS: progression-free survival, TTP: time to disease progression, QOL: quality of life, AEs: adverse events, CRT: chemoradiation, EFS: event-free survival, MTD: maximum tolerated dose, DLT: dose-limiting toxicities.

## *2.3. Existing Skepticism*

The TFF device has been described as "polarizing" amongst neuro-oncology experts. To date, the TTF device is not covered by Medicare or many other insurance companies, on the grounds that the therapy is still experimental. More recently, Novocure has applied for a reconsideration request, which has been accepted for local coverage determination [15].

Despite FDA-approval in the United States (US), existing skepticism regarding the use of the TTF device persists. Primary criticism includes the unblinded nature of the TTF clinical trials. A "sham" device—to better discern a potential placebo-effect of wearing the device—wasn't used. The lack of blinding becomes more of an issue when the primary outcome is PFS. Other concerns arise from the lack of understanding of the TTF device's manner of operation, specifically across a variety of tissues and in combination with other treatments [23]. Furthermore, the fact that randomization in the EF-14 trial occurred over two months after diagnosis suggests a selection bias of patients who did not have progression after the initial treatment and would therefore likely have better survival regardless. Hence, it is difficult to consider the device as "standard-of-care" for future patients who may either: (1) be receiving a shorter course of radiotherapy; or (2) experience an interruption chemotherapy during initial treatment [23].

Several of these points were addressed during a round table discussion of the EF-14 trial at ASCO 2015. This meeting included 5 neuro-oncology experts who did not participate in the trial [21]. In response to the time of randomization question, the EF-14 trial compared favorably in efficacy to other GBM trials when adjustments were made for this difference. Specifically, the improvement in the 2-year survival rate, which was 43% with TTF vs. 29% without TTF, along with median overall survival, (19.6 months with TTF device treatment vs. 16.6 months without TTF device treatment, HR = 0.744, *p* = 0.0038) were considered clinically meaningful [21]. Omission of a placebo-control device in the trial was also a topic of consideration. Several arguments were made in support of this decision, including that: (1) the panel thought it was unlikely that an objective endpoint like OS would be a result of placebo effect; (2) the magnitude of benefit was beyond what would be expected for a placebo effect (HR of 0.75 for OS); and (3) previous trials that lacked a placebo, such as RTOG 0525, did not show improved survival [21]. Ultimately, the group decided that that the TTF device should be considered a treatment option for patients with GBM who were willing to undergo therapy and did not have contraindications. Furthermore, future studies were recommended to identify which subset of patients benefits most from TTF therapy [21].

Despite criticism, most medical device evaluations traditionally lack randomized control groups [24]. Although this may be attributed to the lesser sophistication of clinical trial design by device manufacturers, it's also likely secondary to properties of the device. In this case, patients sense heat from the TTF device, which would be impractical for a sham device. Furthermore, ethical issues arise. For example, it would be difficult and unethical to observe a placebo heart valve replacement [24]. Similarly, it is argued that having a patient shave their head and wear a sham cap for greater than eighteen hours a day would also be unethical. Nevertheless, long-term observational studies will be necessary to observe device efficacy.

#### *2.4. Safety*

Use of a TFF device was not associated with systemic toxicity in either the EF-11 or EF-14 trials. In the EF-11 trial, typical systemic side effects were not seen; whereas in the EF-14 trial, systemic side effect rates did not show a significant difference from the TMZ-alone cohort (48% vs. 44%) [7,13]. Some events in EF-14 had a slightly higher incidence in the TTF group, which was attributed to longer use of TMZ in this group due to delayed progression [7]. A common toxicity was a moderate skin reaction on the scalp below the transducers; dermatitis was observed in 16% of patients wearing the TFF device on EF-11 and 52% of patients in EF-14 [7,13]. Severe skin toxicity was seen in 2% of patients on EF-14 [7]. TFF therapy did not increase the incidence of seizures in either study. The mild to moderate skin reaction patients may experience is reversible and does not require treatment discontinuation [25]. Recently, a phase I clinical trial of 10 patients exploring TTF device treatment with concomitant radiation therapy reported preliminary results of similar rate of TTF-related skin toxicity (40%) to that of the EF-14 trial [26].

## *2.5. Compliance and Quality of Life*

The efficacy of the TFF device has been correlated with patient compliance, and a compliance rate of over 75% has been linked to higher OS [14]. A recently published subgroup analysis showed that a threshold value of 50% compliance, defined as percent usage per month, improved PFS (HR = 0.70) and OS (HR 0.67) [27]. This was seen independent of gender, extent of resection, *MGMT* status, age or performance status [27]. Interestingly, patients with a compliance rate of >90% showed a prolonged median survival of 24.9 months and a five-year survival rate of 29.3% [27].

Consequently, patients need to wear the TFF device continuously with minimal interruption. This inevitably requires lifestyle modifications. Enrollment in the trial was self-selecting and the cohort was not representative of the entire GBM population. Some patients may find the device confining, as it potentially interferes with daily activities. Despite improved survival in the EF-14 trial, patients in clinic may be reluctant to use the TFF device for social or cultural reasons [22]. Others may be averse to shaving their heads. Studies on chemotherapy-induced alopecia have shown that the psychosocial impact of hair loss can be devastating [28]. Quality of life is certainly a priority for this patient population.

A planned interim analysis of the EF-14 trial analyzing health-related quality of life (HRQoL) was performed on 315 patients. HRQoL was slightly improved for patients in the TTF group at six months; physical and social functioning showed no difference at nine and twelve month time points. Patients in the TTF group complained of itchy skin. There was no preliminary evidence that HRQoL, functional status, or cognitive functioning was altered by use of TTF [29]. A final analysis, published in 2018, found that HRQoL did not differ significantly between the two arms of the trial with the exception of itchy skin in the TTF arm [29]. The analysis found that deterioration-free survival was longer in patients receiving TTF therapy for global health, physical and emotional functioning [29]. However, it is important to note that adherence to the HRQoL survey questionnaire decreased significantly over time; survey completion-rate was 91.9% at baseline, 65.8% at three months, and 41.7% at twelve months [29]. Remaining HRQoL scales not previously reported were subjected to an exploratory analysis, which was presented at ASTRO 2018. This study reported a larger proportion of patients having stable/improved bladder control and diarrhea when compared to baseline. The deterioration-free survival for diarrhea, future uncertainty, and headaches was delayed in patients receiving TTF and TMZ treatment when compared to treatment consisting of TMZ alone (HR 0.68, *p* < 0.001). No negative impact on HRQoL was seen in this study [30].

Efforts are underway to improve convenience of utilizing the TTF device. The first-generation Optune TTF device weighed 6 pounds and consisted of a field generator, transducers, lithium batteries and a carry bag. In 2016, a second-generation TFF device weighing only 2.7 pounds was approved [31]. The goal of the second-generation TFF device was to improve patient convenience and compliance. Additionally, the newer device contains an objective log that records treatments compliance [31]. There is hope that the growing popularity of wearable devices, such activity trackers and other smart devices, may encourage patients to be more open about wearing the TTF device [22].

## *2.6. Cost of the Device*

As mentioned previously, the specific payments for this device have not yet been established by Medicare and other insurance providers. Medicare's Ambulatory Payment Classification covers the technical component of the device; Medicare's Physician Fee reimburses the professional component of therapy [29]. The total monthly therapy cost is about \$21,000, which translates to about \$86,000 for the average patient using the device for 4.1 months [30]. Novocure's executive chairman William F. Doyle reported that the company had been providing the device for free for to patients without health insurance in a 2015 New York Times Article [32].

There have been few studies evaluating the cost-effectiveness of TTF. A French group measured the cost-effectiveness of the device and measured it to be "far beyond conventional thresholds due to the prohibitive announced cost of the device." [33]. This report suggested that there is 0% chance of achieving the cost-effectiveness threshold, which was chosen arbitrarily, of €100,000 (\$114,213 in US dollars) per year life gained [33]. Total cost for TTF therapy and conventional therapy strategies were €243,141 and €57,665, respectively. After applying a 4% annual discount according to French national guidelines, the analysis resulted in a total incremental-cost effectiveness ratio of €596 411 per year life gained [33]. A US group presented an abstract at the America Association for Cancer Research (AACR) meeting in 2018 projected that TFF plus TMZ compared to TMZ alone resulted in an undiscounted mean survival of 1.8 life years based on 5 survival results of EF-14 trial [34]. The incremental cost effectiveness ratio based on this survival was \$150,638 per life year gained and \$198,032 per quality life year gained. This abstract reported a high probability of cost-effectiveness at a threshold of \$200,000 [34]. Regardless, the device is certainly expensive and the cost-effectiveness threshold will likely vary considerably between, and even within, countries [35].

How does TTF therapy compare to other novel cancer therapies available in the US? The CheckMate 067 trial evaluating dual checkpoint inhibitors nivolumab and ipilimumab in patients with advanced melanoma showed an increase in PFS from 6.9 months with nivolumab alone to 11.5 months with combination therapy [36]. In 2015, the estimated cost per patient per year for nivolumab was \$103,220 and ipilimumab was \$158,252. The combination therapy was estimated to run \$295,566 [37]. The cost-effectiveness of nivolumab in patients with advanced renal cell carcinoma treated in the US found this treatment to have a 91.7% probability of being cost-effective at a \$150,000 threshold per quality adjusted life year. Other treatments like chimeric antigen receptor (CAR) T-cell therapy are costly with a price tag of \$475,000 for Tisagenlecleucel and \$373,000 for Axicabtagene ciloleucel [38].

Currently, the average direct cost of therapy for GBM patients in the US is about \$8500 per month, and there is regional variability [39]. Mayo clinic reported a median total direct cost of \$91,000 from 1987 to 1992, before the routine use of TMZ: radiotherapy and imaging cost contributed the most to this number [39,40]. It has since been reported that the addition of TMZ, in both the concomitant and adjuvant settings, has led to an eightfold increase to the direct cost of GBM therapy [39,41]. Nevertheless, administration of TTF therapy significantly increases the overall cost of GBM treatment.

### **3. Discussion**

Since the publication of the Stupp trial in 2005, there have been minimal advancements in GBM therapy. Trials that studied targeted molecular inhibitors, immunotherapy agents, vascular growth factor inhibitors, and radiotherapy dose escalation have fallen short. The EF-11 and EF-14 trials have demonstrated that TTF therapy is a promising alternative to chemotherapy in patients with recurrent GBM and adjuvant therapy for patients with newly diagnosed GBM. Most interestingly, the EF-14 trial reported an improved median overall survival of 20.9 months in the TTF plus TMZ group vs. 16.0 months in the TMZ only group. This led to the NCCN endorsement of routine adaptation of TTF as an upfront therapy for patients with newly diagnosed GBM. Since clinical implementation, there have been several reviews on TTF therapy for GBM [21,42]. Given the amount of newer studies done within the last year on this topic, especially in the realm of quality-of-life and cost-effectiveness, we conducted an updated review.

The implementation of the device has polarized many neuro-oncology experts. Some are enthusiastic about this development while others remain skeptical, perhaps understandably. There is a general lack of understanding of the TTF device's mechanism of action beyond what is proposed at the cellular level. Of note, most of the pre-clinical data used to drive the trials was pioneered by Novocure scientists, like Dr. Eilon Kirson. Additionally, the absence of a placebo-control "sham" device in the trials makes some experts less eager to recommend the therapy to their patients. However, it has been discussed that the inclusion of such a device in the trial would have created an ethical dilemma. Moreover, for better or worse, a more lenient precedence for medical devices exists: these devices become approved without randomized, placebo-controlled studies. Again, it is noted that both the EF-11 and EF-14 trials were sponsored by Novocure. Despite these considerations, the EF-14 trial certainly produced compelling results and clinicians echo the phrase "it is difficult to argue with overall survival."

One of the attractive properties of TTF therapy is the minimally-invasive nature and lack of systemic side-effects. This is especially important in the realm of recurrent disease, where patients undergo a variety of treatments from chemotherapy to additional surgery and/or re-irradiation. There are lifestyle drawbacks, as the device needs to be worn as continuously as possible, especially given the recent analyses showing correlation between device compliance and OS. Device compliance of >90% notably correlated with a 5-year OS reaching 30%. It is reassuring that most of the quality of life studies have reported similar results between patients who received TTF and those who did not. Neither health-related quality of life, functional status, nor cognitive functioning have been shown to be altered by TTF use.

The cost of therapy continues to be high, which may be prohibitive for certain patients. Willingness-to-pay for the device varies significantly between countries and even between regions. However, it is important to consider that other novel cancer therapies—such as dual checkpoint blockade with nivolumab and ipilimumab and CAR T-cell therapy—also carry a very high price tag. Hopefully prices will become more manageable as these therapies mature and become more widespread. It's important to make prices manageable for patients while also maintaining incentive for product innovation.

An additional obstacle is that the device is not routinely available in many centers. An electronic survey sent to an international group of radiation oncologists, neurosurgeons, and neuro-oncologists between January 2015 and July 2015 found that only 41% of surveyed practitioners had TTF available to offer their patients [43].

## **4. Conclusions**

Ultimately, long-term, real-world results are necessary to make clinicians more comfortable with the device. Furthermore, positive clinical trial results in disease sites other than GBM may make the device more acceptable and familiar. It remains important to define which subset of patients benefit the most from TTF therapy, through identifying markers that predict improved outcome with TTF therapy. Reluctant patients may be more willing to try the device if it is more convenient and less intrusive to daily life. Further industry efforts to improve the device will help with patient compliance, such as the lower weight in second generation devices. Finally, it is important to define the benefits and potential toxicities of TFF therapy in combination with radiation therapy and chemoradiation therapy via larger, randomized clinical trials. Should we expect to hear much more about TTF therapy in oncology? We'll bet our best hat.

**Author Contributions:** Conceptualization, D.F. and J.D.P.; methodology, D.F.; investigation, D.F.; data curation, D.F. and N.S.; writing—original draft preparation, D.F., M.d.P.G.P.E., I.A., P.G., and J.D.P.; writing—review and editing, D.F., M.d.P.G.P.E., I.A., N.S., J.G., V.P., P.G., and J.D.P.; visualization, D.F., M.d.P.G.P.E., I.A.; supervision, J.D.P.; project administration, J.D.P.

**Funding:** This research received no external funding.

**Acknowledgments:** The Ohio State University Department of Radiation Oncology and The Ohio State University Department of Neuro-Oncology.

**Conflicts of Interest:** The authors declare no conflict of interest.

## **References**

© 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

## *Perspective* **Finding the Right Way to Target EGFR in Glioblastomas; Lessons from Lung Adenocarcinomas**

## **Ya Gao , Wies R. Vallentgoed and Pim J. French \***

Department of Neurology, Erasmus MC Cancer Institute; 3015 CD Rotterdam, The Netherlands<EMAIL_ADDRESS>(Y.G<EMAIL_ADDRESS>(W.R.V.)

**\*** Correspondence<EMAIL_ADDRESS>Tel.: +31-10-704-4333

Received: 9 November 2018; Accepted: 30 November 2018; Published: 4 December 2018

**Abstract:** The *EGFR* gene is one of the most frequently mutated and/or amplified gene both in lung adenocarcinomas (LUAD) and in glioblastomas (GBMs). Although both tumor types depend on the mutation for growth, clinical benefit of EGFR tyrosine kinase inhibitors (TKIs) has only been observed in LUAD patients and, thus-far, not in GBM patients. Also in LUAD patients however, responses are restricted to specific *EGFR* mutations only and these 'TKI-sensitive' mutations hardly occur in GBMs. This argues for mutation-specific (as opposed to tumor-type specific) responses to EGFR-TKIs. We here discuss potential reasons for the differences in mutation spectrum and highlight recent evidence for specific functions of different *EGFR* mutations. These mutation-specific effects likely underlie the differential treatment response between LUAD and GBMs and provide new insights into how to target EGFR in GBM patients.

**Keywords:** EGFR; glioblastoma; glioma; pulmonary adenocarcinoma; lung cancer; erlotinib; gefitinib; lapatinib

## **1. Introduction**

More than fifty years ago, Stanley Cohen reported on the isolation of a heat-stable protein from mouse salivary glands that was able to induce the eruption of incisors and separation of the eyelids [1]. Later studies showed that the eyelid separation was actually a consequence of enhanced keratinization and growth of the epidermis and, based on this function, the protein was coined epidermal growth factor (EGF) [2,3]. Around a decade later, the same group found evidence for a receptor for the growth factor on human fibroblasts and another decade later, the receptor was cloned and sequenced [4,5].

The *EGF* receptor (*EGFR*) gene encodes for a protein of 1210 amino acids which is built up of an extracellular ligand binding domain, a single transmembrane domain, a juxtamembrane region, an intracellular tyrosine kinase domain and a C-terminal regulatory domain. The extracellular region can be divided into four subdomains, domains I–IV, that contain two leucine rich regions (I and III, also known as L1 and L2) and two cysteine rich domains (II and IV, also known as CR1 and CR2). Proteins with a similar domain structure include ERBB2, ERBB3 and ERBB4 (which are also known as HER2-4), and are collectively known as the ERBB protein family, which are all members of the receptor tyrosine kinase superfamily.

In the absence of ligand, EGFR exists as a monomer (or as an inactive dimer) on the plasma membrane. When ligands are available, they bind to the extracellular leucine rich domains I and III, and this association results in the exposure of the cysteine rich domain II, which allows for receptor dimerization. The dimerization then induces phosphorylation of tyrosine residues on the C-terminal intracellular domain of the dimerization partner (trans-phosphorylation) [6]. Phosphorylation occurs on multiple sites: the intracellular domain contains twenty tyrosine residues, twelve of which can be phosphorylated following ligand binding [7]. This phosphorylation results in the recruitment of

specific adaptor proteins and subsequent activation of signal transduction cascades. These cascades include the RAS-RAF-MEK-ERK, PI3kinase-AKT-mTOR, SRC, JNK, PLC-γ-PKC and STAT pathways and ultimately result in cellular proliferation, migration and survival [8,9].

Although the EGF receptor (EGFR) was identified based on its affinity for EGF, later studies showed it can be also activated by six other, related- ligands: heparin-binding EGF-like growth factor (HB-EGF), amphiregulin, epiregulin, epigen, betacellulin and transforming growth factor-alpha (TGF-α) [10]. EGF, TGF-α, HB-EGF and betacellulin are the ligands that bind with high affinity to EGFR; amphiregulin, epiregulin and epigen bind with 10–100 fold less affinity. Each ligand can induce quantitative differences in responses, but can also elicit ligand-specific responses [10,11].

## **2.** *EGFR* **Mutations in LUAD and Gliomas**