Spontaneous Volunteers in Emergencies and Disasters

*Mustafa Yükseler and Jale Yazgan*

### **Abstract**

Spontaneous volunteers are often involved in response and relief efforts during and after emergencies and disasters. These volunteers perform various tasks: search and rescue, first aid, response to fires, distributing food and water, cleaning debris, and caring for animals. It is clear that spontaneous volunteers have played a vital role in preventing the loss of life and property following emergencies and disasters in the past. However, the lack of organization and knowledge about emergencies and disasters among these volunteers creates challenges and risks like liability and security issues. These factors make professional responders reluctant to use spontaneous volunteers in emergencies and disasters. By completing a literature review on the benefits, challenges, and risks of spontaneous volunteers in emergency and disaster response and relief activities, we aim that this study will speed up the fundamental studies on the topic. Creating strategies for these volunteers' efficient usage and incorporating them into the current emergency and disaster management systems can significantly reduce disaster risks.

**Keywords:** emergency, disaster, volunteer, spontaneous volunteer, resilience

### **1. Introduction**

The frequency and effects of disasters are increasing globally. This increase parallels the increase in human–environment interaction, the number of persons exposed to hazards, and the increase in asset values. As of the 21st century, natural and man-made disasters such as September 1, 2001 attacks, Hurricane Katrina, the 2010 Haiti earthquake, the 2011 East Japan earthquake, the Syria Crisis, and the COVID-19 pandemic and climate migration, climate refugee, flood, drought, and desertification caused by climate change have had a global impact [1]. According to a report published in 2020 by the United Nations Office for Disaster Risk Reduction, there have been 7348 natural disasters in the past 20 years (2000–2019), and as a result of these disasters, approximately 1.23 million people have died, 4.2 billion people have been affected, and 2.97 trillion US dollars have been lost to global economic losses. According to the same report, disasters have become far more frequent and severe than they were in the past 20 years (1980–1999) (while 4212 natural disasters occurred, 1.19 million people died, 3.25 billion people were affected, and 1.63 trillion economic losses were caused globally) [2]. Emergency and disaster authorities worldwide and scientists in the field of emergency and disaster place a strong emphasis on

the disaster risk management approach, in contrast to crisis management-oriented disaster management approaches [3–6]. Making societies disaster-resistant is a crucial component of disaster risk management, in addition to reducing structural risks. There are many instances where emergencies and disasters outstrip a nation's response capacity. Volunteering is thus one of the key initiatives for enhancing societal resilience to disasters [3, 4, 7, 8].

Volunteering plays a significant role in many countries' response and recovery efforts during emergencies and disasters. When a disaster strikes, volunteers help in various fields, such as technical rescue, fire response, medical response, debris cleaning, logistical support, and transportation [8]. According to the World's Volunteerism Report, millions of volunteers participate in activities across a range of sectors, and their economic impact is equivalent to almost 61 million full-time employees per month based on the number of full-time employees [9]. These statistics show that volunteering activities are important for achieving sustainable development goals.

Volunteering activities in terms of emergencies and disasters are carried out in two ways as formal and informal [10]. The activities of the Red Cross or the Red Crescent organizations can be examples of formal volunteering. On the other hand, informal volunteering is when people go to the affected area voluntarily and engage in response and relief activities after an emergency or disaster event, called *"convergence reaction"* [11] in the literature. This has been happening instinctively since the existence of human beings. These volunteers who participate in emergency and disaster response and relief activities are referred to by several terms in the literature, including spontaneous volunteers, unorganized volunteers, unaffiliated volunteers, or informal volunteers [12, 13]. These volunteers are referred to in this study as "spontaneous volunteers". Various definitions of spontaneous volunteers can be found in the literature. Generally, they are defined as: *"spontaneous volunteers are individuals or groups of people who seek or are invited to contribute their assistance during and/or after an event, and who are unaffiliated with any part of the existing official emergency management response and recovery system and may or may not have relevant training, skills or experience"* [14]. During past emergencies and disasters, it can be argued that these volunteers performed crucial roles. Below are some of the disasters that spontaneous volunteers participated in:

Although spontaneous volunteers participate in various activities and make financial contributions during emergencies and disasters, their lack of training and skills in response and recovery activities might provide obstacles rather than benefits. Alexander [8] defined the reasons for this situation as illustrated in **Figure 1**.

According to the historical experiences above, spontaneous volunteers have significantly contributed to response and recovery studies, both qualitatively and quantitatively. For the reasons outlined in **Figure 1**, these volunteers nevertheless posed threats to not only the affected individuals but also the professional responders in the same disaster events. For instance, it was determined that 2.2 rescuers per person pulled alive from the debris in the 1985 Mexico City earthquake died because they lacked the necessary skills, knowledge, and tools for urban search and rescue [30]. Similar obstacles prevented emergency response teams from reaching the scene following the 1999 Turkey Marmara earthquake because spontaneous volunteers caused a 32-kilometer traffic jam [24, 30].

#### **Figure 1.**

*Barriers that unorganized volunteers may create in emergencies and disasters [8].*

The function of spontaneous volunteers in emergencies and disasters is discussed in this paper. The study aims to advance fundamental research on spontaneous volunteerism in emergencies and disasters. The study is divided into the following sections: volunteers as human resources in emergencies and disasters, spontaneous volunteers, benefits of spontaneous volunteers in emergencies and disasters, challenges and risks of spontaneous volunteers in emergencies and disasters, and conclusion.


*4.1.2. Melt mixing*

For mixing in solution, the polymer matrix must be soluble in at least one solvent. This is problematic for many polymers. Melt mixing is generally applicable and fairly simple, especially when used in the case of thermoplastic polymers. In the melt spinning process, carbon nanotubes are mechanically dispersed in the polymer matrix using a high-shear mixer at high temperature [26]. This approach is simple and compatible with existing industrial technologies. Shear forces destroy CNT aggregates and prevent their formation.

The disadvantage of this method is that this method produces a dispersion of carbon nanotubes in the polymer matrix, which is significantly worse than the dispersion that can be achieved by mixing in solution. In addition, carbon nanotubes should be smaller because of the high viscosity of the composites with a higher content of carbon nanotubes.

#### *4.1.3. In-situ polymerization*

Using this method, carbon nanotube or nanostructured silicon carbide is dispersed in the monomer followed by polymerization. Moreover, a higher percentage of fillers can be easily dispersed, and they form a strong interaction with the polymer of the matrix. This method is used for the preparation of composites with polymers that cannot be processed by mixing in solution or melt mixing, for example insoluble and thermally unstable polymers.

In the case of using a polyimide matrix, which is obtained by polycondensation, it is expedient to use the in-situ polymerization method.


**11. Conclusions**

The problem of HCW management in general and hazardous and nonbiodegradable portion thereof is too big to be ignored or else it will boomerang into something, which will be very difficult to manage. Though, at first sight, it does not look that severe a close analysis reveals that it is one of the major sources of greenhouse gas emission and comprehensively affects all the components of the environment and contributes to global warming. One of the serious aspects of HCW is its threat to the health of humans and animals as a good part of HCW contains infectious waste. The HCW contaminate the air, water, and soil of the surrounding environment releasing toxic substance like chemicals including heavy metals and radioactive substances, carcinogenic toxic gases like dioxin and furan, and causing many health issues not only in humans but also in animal species. The surrounding areas in dumping and processing sites are affected by these pollutants making them inhabitable. There is an urgent need for comprehensive research on the health hazards of HCW and measures to prevent them, processing of HCW and development of biomarkers for risk assessment of HCW, and a standard protocol for its processing that can be made mandatory to be followed by HCI. Unless there are universal consensus and framework for dealing with HCW the problem will not be solved. The healthcare industry needs to adopt eco-friendly approaches and operations and initiate whatever it takes to really become a health care provider and not itself become a threat to the health of living and nonliving environment by indiscriminate toxic HCW generation. Measures like extensive awareness and training programs, setting up environment-friendly hospitals, and avoiding single-use materials should be undertaken. Refuse, reduce, reuse, recycle, recover, and dispose should be the governing principles of HCW management. With collective responsibility, sincerity and honest efforts the menace of HCW in general and nonbiodegradable in specific can be not only controlled but contained for good of the environment in general and human health in specific.

*The Toxicity of Environmental Pollutants*


*Review* **Childhood Malignant Brain Tumors: Balancing the Bench and Bedside**

**Colin Thorbinson 1 and John-Paul Kilday 1,2,\***

**Simple Summary:** Brain tumors remain the most common childhood solid tumors, accounting for approximately 25% of all pediatric cancers. They also represent the most common cause of cancer-related illness and death in this age group. Recent years have witnessed an evolution in our understanding of the biological underpinnings of many childhood brain tumors, potentially improving survival through both improved risk group allocation for patients to provide appropriate treatment intensity, and novel therapeutic breakthroughs. This review aims to summarize the molecular landscape, current trial-based standards of care, novel treatments being explored and future challenges for the three most common childhood malignant brain tumors—medulloblastomas, high-grade gliomas and ependymomas.

**Abstract:** Brain tumors are the leading cause of childhood cancer deaths in developed countries. They also represent the most common solid tumor in this age group, accounting for approximately one-quarter of all pediatric cancers. Developments in neuro-imaging, neurosurgical techniques, adjuvant therapy and supportive care have improved survival rates for certain tumors, allowing a future focus on optimizing cure, whilst minimizing long-term adverse effects. Recent times have witnessed a rapid evolution in the molecular characterization of several of the common pediatric brain tumors, allowing unique clinical and biological patient subgroups to be identified. However, a resulting paradigm shift in both translational therapy and subsequent survival for many of these tumors remains elusive, while recurrence remains a great clinical challenge. This review will provide an insight into the key molecular developments and global co-operative trial results for the most common malignant pediatric brain tumors (medulloblastoma, high-grade gliomas and ependymoma), highlighting potential future directions for management, including novel therapeutic options, and critical challenges that remain unsolved.

**Keywords:** pediatric; brain; tumor; medulloblastoma; glioma; ependymoma

### **1. Introduction**

Brain tumors are the most common solid tumors of childhood, accounting for approximately 25% of all pediatric malignancies, and represent the leading cause of cancer-induced morbidity and mortality in this age group [1]. With an incidence of approximately 6 per 100,000 children in industrialized society [2], these tumors represent a spectrum of clinically, pathologically and biologically diverse subtypes which can pose significant challenges in conducting research and clinical trials, necessitating international collaboration.

Over recent decades, cure rates for selected pediatric brain tumors (most notably medulloblastoma) have improved [3], predominantly as a consequence of advances in multiparametric neuro-imaging, neurosurgical techniques, radiation therapy and multiagent chemotherapy, together with improved supportive care. However, such survival

**Citation:** Thorbinson, C.; Kilday, J.-P. Childhood Malignant Brain Tumors: Balancing the Bench and Bedside. *Cancers* **2021**, *13*, 6099. https:// doi.org/10.3390/cancers13236099

Academic Editors: Saurabh Agarwal and Jianhua Yang

Received: 8 November 2021 Accepted: 29 November 2021 Published: 3 December 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 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 (https:// creativecommons.org/licenses/by/ 4.0/).

advances are typically offset by a therapy-induced toxicity burden for the patient, with wide-reaching consequences for the child, their family and society. Moreover, for the majority of brain tumors, prognosis has remained static for over 30 years despite these technological improvements.

To overcome this impasse, the pediatric neuro-oncology community has shifted focus to develop risk-stratified treatment protocols that aim to reduce iatrogenic morbidity while maintaining outcomes for favorable-risk lesions, and improve cure rates for tumors refractory to conventional therapy, either through intensification or novel agents. This strategy has been supplemented by an evolution in our understanding of the molecular pathogenesis of almost all pediatric brain tumors.

Such molecular advances have identified potential cells of origin, and led to the identification of multiple biologically distinct subgroups within most brain tumor entities, therein allowing accurate risk stratification for affected children when incorporated with clinical, histological and survival data. In addition, oncogenic biological pathways amenable to manipulation using novel targeted agents have been identified.

This article will provide a summary of the most common malignant pediatric brain tumors (medulloblastoma, high-grade gliomas and ependymoma) with particular focus on inherent molecular advancements and potential future directions for management, including novel therapeutic options.

#### **2. Medulloblastoma**

#### *2.1. Background*

Medulloblastoma (MB) represents the most common malignant brain tumor in children, accounting for approximately 20% of all central nervous system (CNS) tumors [2,4]. It also comprises over 60% of intracranial embryonal tumors, a recently characterized entity consisting of atypical teratoid rhabdoid tumors (ATRTs), embryonal tumors with multilayer rosettes (ETMRs), CNS neuroblastoma with *FOX2* alteration and malignant neuroepithelial tumors with *BCOR* alteration [5].

Arising within the cerebellum, MBs are observed across all age categories but are most frequently identified at a median age of five years [6]. Demographic, histological and prognostic heterogeneity embody MB, while it represents the first brain tumor where revolutionary global initiatives (such as the Medulloblastoma Advanced Genomics International Consortium (MAGIC)) have transformed our understanding of the molecular underpinnings of MB pathogenesis, enabling improved patient risk stratification to potentially influence clinical outcome [7].

#### *2.2. Histopathology*

MBs share a primitive embryonal phenotype comprising malignant cells of stereotypic histological patterns, dominated by neuronal antigen expression [8]. World Health Organization (WHO) pathological classification systems have historically divided MB into a classic subtype accounting for 72% of all cases, a desmoplastic/nodular variant of which medulloblastoma with extensive nodularity (MBEN) is a subgroup and a large cell/anaplastic variant which has historically been assigned an adverse prognostic association [5,9].

#### *2.3. Molecular Classification*

In the past decade, seminal transcriptomic MB analyses led to a global consensus establishing the identification of four discrete molecular subgroups, likely arising from distinct cells of origin—wingless-activated (WNT), sonic hedgehog (SHH), Group 3 and Group 4 MB [10,11]. Further molecular scrutiny of these four groups has now identified somatic mutations targeting chromatin modification as the leading driver for MB heterogeneity via epigenetic dysregulation [12]; further subdivisions have now been established [13–16] (Figure 1).

*Cancers* **2021**, *13*, x 4 of 28

#### 2.3.1. WNT Activated (WNT)

WNT MBs account for approximately 10% of all MBs, and often arise in older children with equal gender distribution [11]. Typically occurring in the midline, they frequently invade the lateral recess of the brainstem through the foramen of Luschka, due to a lower rhombic lip cell of origin [17,18]. They rarely metastasize and morphology is typically of the classic variant [8].

Somatic activating mutations in exon 3 of *CTTNB1*, which encodes B-catenin, are found in 80–90% of WNT MB, with 85–90% displaying monosomy 6 [19–23]. Mutations in the adenomatous polyposis coli (*APC*) gene are common in WNT tumors lacking *CTTNB1* mutations [15,24]. Less frequently occurring mutations include *TP53*, *SMARCA4*, *KMT2D* and *DDX3X* [11,15,25,26]. *TP53* mutation occurs only in a minority of WNT MB, and is not prognostic, unlike the SHH subtype [27].

#### 2.3.2. Sonic Hedgehog-Activated-Activated (SHH)

SHH MB represents approximately 30% of all cases, presenting predominantly in a bimodal age distribution; below three years and in young adults [5,8,10]. Originating from granule progenitor cells SHH MBs localize almost exclusively within cerebellar hemispheres [17,28]. All nodular desmoplastic MBs belong to the SHH subgroup, although other histologies can be observed [21,29]. They are most commonly localized at diagnosis and morphology frequently correlates with underlying genetic abnormalities.

SHH MBs are characterized by activation of the SHH pathway as a result of somatic or germline mutations in a number of genes including *SMO*, *PTCH1* and *SUFU* [30]. While *PTCH1* mutations are seen across 30–50% of SHH MBs, *SUFU* and *SMO* mutations are typically seen in infant and adult SHH MBs, respectively [30]. *TP53* mutations typically arise in childhood SHH MBs [27]. Recent epigenomic profiling has identified a further four clinically distinct granular molecular subclasses of SHH MB, alpha, beta, gamma and delta [13]. SHH-alpha MBs predominate in children, whereas infants are most commonly associated with SHH-beta and SHH-gamma, and SHH-delta is typically observed in adult patients [8].

#### 2.3.3. Group 3

Group 3 tumors account for 25% of all MB cases, predominate in males and occur most frequently in younger children between the ages of 2 and 5 years [8]. Thought to arise from neural stem cell origin [31], Group 3 MBs have a short symptom interval and are frequently metastatic at diagnosis with small primary tumors [11,28,32].

As with Group 4 MB, Group 3 tumors are not characterized by a signature oncogenic pathway. Nevertheless, Group 3 MBs can be associated with activation of GABAergic and photoreceptor pathways [33,34]. Broad genomic aberrations are a feature, while recurrent somatic nucleotide variants are infrequent [7,12,26]. *MYC* amplification is the most common finding (in approximately 17% of cases) commonly occurring within a complex chromosomal rearrangement at the 8q24 locus, resulting in *MYC*–*PVT1* fusion [7,12,13,34]. The presence of isochromosome 17q, activation of growth factor proto-oncogenes *GFI1* and *GFI1B*, and amplification of transcription factor *OTX2* are also observed [13,15,35].

#### 2.3.4. Group 4

Group 4 tumors represent 35% of all MBs, have a male predisposition and are the dominant molecular subgroup in children of 3 to 16 years of age [8,36]. Similarly to Group 3 MB, they arise in the fourth ventricle and are frequently metastatic at diagnosis, but have a longer symptom interval [11,32].

Genetic abnormalities seen in Group 4 tumors include inactivating mutations of the histone demethylase *KDMS6A* and histone modulator *PRDM6*, tandem duplications of *SNCAIP* and amplifications of *CDK6* and *MYCN* [7,12,25,26,33]. Chromosomal copy number variations include deletion of chromosome 8, 11 or 18p, gain of chromosome 1 or 17q and isochromosome 17q, the most common cytogenetic abnormality in the subgroup [37].

#### *2.4. Prognostic Factors*

Typical risk-stratification systems for MB incorporate age, extent of tumor resection, and metastatic status to define standard and high-risk cohorts, in turn determining therapy administered. Standard-risk patients are older than 3 years, have undergone gross or near total excision (below 1.5 cm2 of residual tumor) with localized disease while remaining patients are classified as high risk. However, these and historical prognostic markers (such as anaplastic morphology) may indeed be surrogates for the underlying MB molecular subgroup, suggesting future stratifications require further refinement.

Pediatric patients with standard-risk WNT-activated MB have an excellent prognosis with a 5 year progression-free survival above 90% following standard therapy. SHH MB demonstrates a range of outcomes. Infant SHH MBs beta and gamma have disparate outcomes, with beta conferring a poor prognosis, and gamma good outcomes [38,39]. *TP53* germline positive SHH MBs confer a poor prognosis with a post-therapy 5 year survival of just 30–40%, particularly when associated with *MYCN* and *GLI2* amplification [40], whereas wildtype SHH MB are associated with a favorable outcome with a 5 year survival of approximately 80% [8,27,30].

Group 3 and 4 MBs also demonstrate variable outcomes, influenced by inherent molecular heterogeneity spanning both groups [14]. For example, Group 3 MB generally carry a poor prognosis, particularly *MYC* amplified cases which are often refractory to conventional therapy [41–43], while Group 4 MBs demonstrate a variable prognosis, incorporating favorable-risk MBs harboring chromosome 11 loss or chromosome 17 gain [14]. Infantile Group 4 MBs are infrequent but carry a poor prognosis [44].

#### *2.5. Current Management/Clinical Trials*

The sequential trial-based addition of adjuvant craniospinal radiotherapy and combination chemotherapy to maximal safe tumor resection has improved survival rates for standard-risk patients immeasurably over the last 50 years and is now the accepted standard of care (Table 1). However, such improved cure rates are achieved at a significant burden to the survivor, with most experiencing chronic neurocognitive and neuroendocrine morbidities [45,46]. While standard-risk patients have benefited from a trial-validated reduction in craniospinal radiotherapy intensity [47] (Table 1), high-risk patients continue to require high-dose radiotherapy (36 Gy) and intensified chemotherapy regimens to maintain a 5 year progression-free survival (PFS) of up to 70% [48,49] (Table 1).

Current trial designs utilize refined patient risk stratifications which incorporate the additional knowledge of molecular MB subgroups. Open standard-risk studies including the Children's Oncology Group (COG) ACNS1422 (NCT02724579), the North American SJMB12 (NCT01878617) and the European SIOP PNET5 trial (NCT02066220) are assessing whether treatment intensity can be reduced without compromising survival rates for favorable-risk MBs (particularly WNT-activated MBs).

**Table 1.**

Multinational

collaborative

 clinical trials in pediatric

medulloblastoma,

 high-grade

 gliomas

 and

ependymoma,

 published

 since 2000.

GTR: gross total resection; DIPG: diffuse intrinsic pontine glioma; yrs: years; mnths: months.

Caution regarding de-escalation of therapy for WNT-activated MBs is evident from the premature termination of trial NCT02212574 which abandoned craniospinal irradiation for these patients, and a recent retrospective analysis of 93 WNT-activated MBs where relapse was associated with a reduction in the cumulative dosing of maintenance chemotherapy [61].

The PNET5 trial is also assessing the radio-sensitizing effect of carboplatin for non-WNT MB, while SJMB12 is the addition of targeted drug therapy in conjunction with conventional agents for specific molecular subgroups (SHH and high-risk Group 3 and 4 MBs). A European biomarker-driven phase III trial for newly stratified high-risk MB opened to recruitment in 2021. Of interest, post-operative residual tumor is not considered a high-risk feature in this study. The trial incorporates a double-randomized design, comparing the efficacy of hyper-fractionated radiotherapy and additional high-dose chemotherapy against standard radiotherapy, followed by a comparison of multimodal continuation chemotherapy versus single agent temozolomide (EudraCT Number: 2018-004250-17).

Infant MB represents a distinct, intensive chemotherapy-only treatment group [29]. Outcomes for infants with nodular desmoplastic SHH MB can be excellent, although it appears that this requires the inclusion of intrathecal methotrexate in addition to systemic therapy [38,39,62,63]. The COG ACNS0334 study of non-nodular desmoplastic MBs, incorporating both induction and high-dose tandem consolidation cycles of chemotherapy reported 100% survival for metastatic SHH MBs and a survival advantage for the incorporation of methotrexate at induction in Group 3MBs [64].

#### *2.6. Novel Therapies*

Advances in molecular understanding of MB pathogenesis have also provided the opportunity for the application of subgroup-specific novel targeted therapeutics, notably for SHH MBs. Vismodegib and sonidegib are *SMO* inhibitors that have shown objective responses in pediatric recurrent SHH MB [65–71]. For most patients, such responses were not sustained, as a result of mutations downstream from *SMO* re-activating the pathway [30]. Another important consideration of this therapy is the association with premature growth plate fusions which has led to modification of the current SJMB12 study [70–72]. Agents such as silmitasertib, targeting *SMO* downstream mutations in the SHH pathway, are under evaluation in relapsed SHH MB (NCT03904862). *GLI* inhibition by arsenic trioxide is another area of drug development in SHH MB and early phase pediatric tumor trial data are awaited (NCT00024258).

For non-SHH tumors, the aforementioned SJMB12 study is evaluating the addition of pemetrexed and gemcitabine to conventional chemotherapeutic agents for high-risk Group 3 and 4 MBs (large cell anaplastic histology, metastatic disease or *MYC*/*MYCN* upregulation) after promising high throughput in vitro drug assay analysis [73]. The CDK4/6-cyclin D-Rb pathway was identified as a potential therapeutic target in xenograft models for non-WNT MB [74]. Other proposed approaches include *HDAC* inhibitors, *PI3K* inhibition and BET-bromodomain inhibition to downregulate *MYC* expression in Group 3 MBs, and *LSD1* inhibition of *GFI1*/*GFI1B* overexpression when present in Group 3 and 4 MBs [75–78].

Finally, despite the challenge posed by the lack of immunogenic targets in CNS tumors, immunotherapy has been proposed as a potential treatment option in relapsed/refractory MB [79]. Anti-EPHA2, HER2 and IL-13Rα2 chimeric antigen receptor T-cell (CAR-T) therapy has been shown to successfully treat murine Group 3 MBs [80] and early phase trials in children have commenced (NCT03500991, NCT04661384).

#### **3. High-Grade Gliomas**

#### *3.1. Background*

This group encapsulates all malignant lesions of glial origin. Alongside embryonal tumors, pediatric high-grade gliomas (pHGGs) are one of the most common malignant tumor groups of the childhood central nervous system, with a collective incidence of 1.1 per 100,000 children [2]. Despite a paradigm shift in our understanding of pHGG molecular subgrouping being distinct from adult counterparts, and some therapeutic successes for particular entities (such as infant HGG), little progress has been made over recent decades to improve the dismal prognosis; pHGGs account for over 40% of all childhood brain tumor deaths [81]. As a result, they remain the focus of several experimental therapeutic research teams.

#### *3.2. Histopathology*

The vast majority of pHGGs can be classified as anaplastic astrocytomas (WHO Grade III), or glioblastoma (Grade IV). Historically, a minority of diffuse intrinsic pontine gliomas (DIPGs) were morphologically consistent with diffuse astrocytoma (Grade II), likely resulting from sampling bias. However, the identification of pathognomonic oncogenic mutations in DIPG (particularly in histones 3.1 and 3.3), together with established malignant clinical characteristics, resulted in an amendment to current WHO nomenclature, with DIPGs now classified as diffuse midline gliomas with *H3K27* mutation (Grade IV) [5].

#### *3.3. Molecular Classification*

Clear biological distinctions between pHGGs and adult counterparts are now established [82,83], providing a rationale for the failure of many novel therapies derived from adult tumor research. Molecular heterogeneity within pHGGs is also well described [84–91]. The largest molecular meta-analysis of pHGGs published to date, incorporating genomic, epigenomic and transcriptomic profiling has now identified at least nine pHGG subgroups with inherent biological and/or clinical characteristics such as age, tumor location and prognosis [90]. These subgroups express recurrent signature aberrations, which may lead to further refinement of subdivisions in the future (Figure 2).

The predominant pHGG subgroups express mutations of histones *HIST1H3B* (*H3.1*) at position K27, *H3.2* (rarely) and *H3F3A* (*H3.3*) at positions K27 and G34 [90,92]. H3K27M pHGGs are characterized biologically by aberrant expression resulting from loss of trimethylation at lysine 27 on Histone 3 [93,94], and clinically by their midline location (pons, midbrain, thalamus, spina cord) and younger patient age [90,91]. H3.3 G34 subgroup pHGGs are typically located in hemispheric locations, impacting adolescent and older age groups [90,91,95]. The midline location may contribute to the significantly poorer prognosis reported in K27 pHGGs versus G34 counterparts [85,90,91,95], although the mutations alone have been reported as independent prognostic markers in multivariate analysis [90]. Secondary aberrations within the pHGG histone subgroups have also been identified. *TOP3A*, *CCND2*, *PDGFRA*, *PPM1D*, *TP53* and *FGFR1* mutations are more frequently identified in H3.3K27 pHGGs, while H3.1K27 tumors often demonstrate *PI3K* and *ACVR1* mutations and H3.3 G34 pHGGs typically contain *TP53* and *ATRX* mutations [90].

Other subgroups include the IDH mutant pHGGs, associated with a frontal location, an adolescent age range and improved prognosis, hypermutant pHGGs as seen in DNA replication repair deficiency disorders, infant HGGs characterized by *NTRK* mutations and pleomorphic xanthoastrocytoma-like pHGGs and *BRAF* mutated pHGGs, which may represent low-grade lesions that have undergone malignant transformation [90,91,95]. The latter two subgroups may be amenable to novel targeted inhibitor agents and often demonstrate good responses to therapy and improved survival outcomes. A final 'wildtype' subgroup comprises pHGGs harboring mutations in genes such as *NF1*, *MYCN*, *EGFR*, and *CDK6* [90].

**Figure 2:** Molecular subgroups of pediatric high-grade glioma. At least nine subgroups are thought to exist, with biological and clinical features highlighted in accordance with [84,90,91]. Agerelated cartoons depict infant, young child (2–5 years), child (5–12 years), and adolescent/adult (12+ years). Key: amp. = amplification, mut. = mutation, del. = deletion, actvn. = activation, IDH = isocitrate dehydrogenase, and PXA, pleomorphic xanthoastrocytoma. **Figure 2.** Molecular subgroups of pediatric high-grade glioma. At least nine subgroups are thought to exist, with biological and clinical features highlighted in accordance with [84,90,91]. Age-related cartoons depict infant, young child (2–5 years), child (5–12 years), and adolescent/adult (12+ years). Key: amp. = amplification, mut. = mutation, del. = deletion, IDH = isocitrate dehydrogenase, and PXA, pleomorphic xanthoastrocytoma.

*Cancers* **2021**, *13*, x 11 of 28

#### *3.4. Prognostic Factors*

Prior to the advent of molecular subclassification as described above, the two leading clinical prognostic factors were the extent of surgical resection and tumor histological grade with incomplete resection and Grade IV HGGs conferring a dismal prognosis [96,97]; this continues to be the case today but is supplemented by molecular stratification also. Some studies have also reported a prognostic influence of methylguanine-DNA-methyltransferase (MGMT) expression in the efficacy of temozolomide therapy and patient outcome [54,98].

#### *3.5. Current Management/Clinical Trials*

The global standard of care for pHGG, the Stupp regimen, stems from adult glioblastoma trial work, which demonstrated that the addition of the alkylating agent temozolomide alongside and after focal radiotherapy, improved progression-free and overall patient survival [99]. Given the molecular disparity between adult HGG and their childhood counterparts, it is therefore unsurprising that temozolomide in a Children's Oncology Group (COG) pHGG trial analysis (ACNS0126) did not improve outcome compared with previous trials using varied adjuvant chemotherapies [54] (Table 1). However, it remains the standard of care because of the relatively low toxicity profile in comparison to alternative regimens.

The COG ACNS0423 trial noted a marginal outcome benefit for the addition of lomustine with temozolomide [55]; however, it was unclear if this was specific to certain molecular subgroups, while the myelosuppressive toxicity of the regime often proved restrictive. The German Hirntumor (HIT) co-operative group have also reported an improved survival rate for a subset of children with glioblastoma achieving gross total resection compared to historical controls, using an intensified chemotherapy regime alongside and after RT [100].

No definitive therapeutic breakthrough has been made in the treatment of DIPG (now diffuse midline glioma, H3K27 mutant), such that the standard therapy remains radiotherapy alone (Table 1). Modern, multinational collaborative trials, such as the Innovative Therapies for Children with Cancer (ITCC) BIOMEDE study, are developing a more nuanced approach alongside focal RT, utilizing novel inhibitor therapy to target corresponding molecular aberrations present in the lesion (dasatanib, everolimus, and erlotinib) (NCT02233049). Interim overall survival analysis of 193/250 randomized patients concluded that a preferential agent was unlikely to be demonstrated, with survival rates comparable with RT alone, albeit everolimus had the most favorable toxicity profile [58].

#### *3.6. Novel Therapies*

The paradigm shift in understanding of the molecular heterogeneity of pHGG, together with the failure of conventional therapeutics to significantly improve outcomes for several years, has shifted focus towards developing novel agents that manipulate the epigenetic and genomic aberrations inherent in pHGG molecular subgroups, immunotherapies, and the development of alternative drug administration routes to penetrate the blood– brain barrier such as convection enhanced delivery for diffuse midline glioma H3K27 mutant/DIPG [92,101–105].

Success of mutational target inhibition in specific pHGG subgroups gives credence to this new therapeutic standpoint. For pHGGs with *BRAF V600E* mutations, BRAF inhibitor (BRAFi) activity has been demonstrated as salvage therapy [106–109]; international cooperative studies are recruiting (NCT03919071). Similar findings of efficacy have been made with neurotrophic tyrosine receptor kinase inhibitor agents for infant HGGs [110,111] and immune checkpoint inhibition in hypermutant pHGGs resulting from replication repair deficiency disorders [112–115]. Follow-up co-operative early phase trials are now open (NCT04267146, NCT04323046 and NCT04655404).

With respect to the other main subgroups, targeting histone modification is a therapeutic research focus for the H3.1–3.3 pHGG subgroup. Histone deacetylase inhibitors (HDACi) such as panobinostat, vorinostat and valproic acid have been postulated to improve the therapeutic landscape for this subgroup following successful HDACi in vitro pHGG studies, but translational results to date have proved disappointing [116–119]. Other agents being looked at for this subgroup include ACVR1/ALK inhibitors [120,121] and the imipridones incorporating agents such as ONC201 [122–124].

For the IDH mutant pHGG subgroup, blood–brain barrier penetrant IDH inhibitors have been developed for glioma trials (NCT02273739, NCT03343197, NCT02073994 and NCT04056910). These may be specific to IDH-1 (ivosedinib), IDH-2 (enasidenib) or both (vorasidenib). In addition, the use of PARP (poly-adenosine 50-diphosphate-ribose) inhibitors alongside temozolomide as a radiosensitizer is being explored [125].

Immunotherapeutic strategies other than checkpoint blockade are also being evaluated in pHGGs, including cancer peptide vaccine therapy with antigens such as Ephrin A2 (EphA2), interleukin 13 receptor alpha 2 (IL13Ra2), survivin and HLA-A2 (NCT01130077) [126–128], autologous dendritic cell vaccine therapy [129], and chimeric antigen receptor (CAR)-T therapy where studies are recruiting (anti-IL13aR2; NCT02208362, anti-GD2; NCT04196413, anti-B7 H3; NCT04185038).

#### **4. Ependymoma**

#### *4.1. Background*

Ependymoma is the second most common malignant brain tumor entity in children, after medulloblastoma, representing approximately 10% of all childhood CNS tumors [130]. Most cases present in patients aged below five years and have a male predominance (male: female ratio 0.23: 0.17) [130,131]. Although able to arise anywhere in the neuraxis, over 90% of pediatric ependymomas are intracranial (IC) in origin. Of these, two-thirds occur in the posterior fossa (PF), with the remaining one-third located in the supratentorial (ST) compartment [132]. Leptomeningeal metastasis is uncommon, reported in 2–20% of cases [133,134].

No inherited disorders are consistently reported to predispose to IC pediatric ependymomas. Neurofibromatosis type 2 appears to be associated with the development of spinal ependymomas but typically in the adult population [135].

#### *4.2. Histopathology*

Current histological classification of ependymoma remains according to the current WHO grading scheme, resulting in four main histological subgroups: subependymoma and myxopapillary ependymoma (grade I), classic (grade II) and anaplastic (grade III) [120]. Subependymoma typically arise in the ventricles of adults, while myxopapillary ependymoma occur exclusively in the spine [5,136]. Consequently, classic and anaplastic variants typically account for all pediatric IC ependymomas. Morphologically they are both characterized by the tumor cell formation into true rosettes (around a canal) or pseudorosettes (around a blood vessel) while anaplasia is signified by increased mitotic figures, necrosis, microvascular proliferation, and an increased an increased cellular nucleus/cytoplasmic ratio [5]. Common immunohistochemical findings include positive staining for glial fibrillary acid protein (GFAP), expression of EMA, S100 and vimentin [5,137].

The utilization of histological grading as a prognostic marker has failed to consistently be of value, in part due to the subjective nature of grade assignation and tumor heterogeneity. These factors, alongside improved understanding of the genomic landscape of pediatric ependymoma, has led to the Consortium to Inform Molecular and Practical Approaches to CNS Tumor Taxonomy (cIMPACT) to recommend that the WHO adopt a new, integrated histological/biological classification system for ependymomas [138].

#### *4.3. Molecular Classification*

Genomic and methylomic profiling of ependymoma has revealed nine distinct molecular subtypes, four of which account for most pediatric IC ependymoma across the PF (PF-A and PF-B) and ST (ST-ZFTA and ST-YAP) compartments [139] (Figure 3).

Genomic and methylomic profiling of ependymoma has revealed nine distinct molecular subtypes, four of which account for most pediatric IC ependymoma across the PF

(PF-A and PF-B) and ST (ST-ZFTA and ST-YAP) compartments [139] (Figure 3).

*4.3. Molecular Classification*

**Figure 3.** Predominant molecular subtypes of pediatric intracranial ependymoma. Posterior fossa and supratentorial childhood ependymomas are shown, further categorized into four in-group subtypes; PF-A, PF-B, *ZFTA*-fused and *YAP1* fused. The clinical and biological characteristics of these subtypes are shown, in accordance with [59,120,139–153]. Nine molecular subtypes of ependymoma are reported but the remaining subtypes occur in either the spinal cord (spinal subependymoma, spinal myxopapillary ependymoma, spinal ependymoma) or the adult brain (subependymoma: PF and ST) so are not depicted in this figure. Age-related cartoons depict infant, young child (2–5 years), child (5–12 years), adolescent/adult (12+ years). Key: WHO = World Health Organization, CIN = chromosomal instability, GTR = gross total resection, IR = incomplete resection. PF-A ependymomas are biologically characterized by epigenetic dysregulation of **Figure 3.** Predominant molecular subtypes of pediatric intracranial ependymoma. Posterior fossa and supratentorial childhood ependymomas are shown, further categorized into four in-group subtypes; PF-A, PF-B, *ZFTA*-fused and *YAP1*-fused. The clinical and biological characteristics of these subtypes are shown, in accordance with [59,120,139–153]. Nine molecular subtypes of ependymoma are reported but the remaining subtypes occur in either the spinal cord (spinal subependymoma, spinal myxopapillary ependymoma, spinal ependymoma) or the adult brain (subependymoma: PF and ST) so are not depicted in this figure. Age-related cartoons depict infant, young child (2–5 years), child (5–12 years), adolescent/adult (12+ years). Key: WHO = World Health Organization, CIN = chromosomal instability, GTR = gross total resection, IR = incomplete resection.

DNA methylation and histone modification, often accompanying lack of *H3K27* trimethyl-

ation [147,149,150]. With the exception of some genomic imbalances, namely 1q gain and 6q loss, they typically demonstrate a balanced genome [139,147,149]. They are most common in infants and young children, have a tendency towards infiltration, dissemination and consequent poor prognosis [153]. Due to their predominant lateral location and inherent invasiveness, gross total resection (GTR) is often difficult to achieve and therefore relapse rates are high [154]. PF-B ependymomas are characteristically enriched with numerous cytogenetic abnormalities and are more common in adolescents and young adults PF-A ependymomas are biologically characterized by epigenetic dysregulation of DNA methylation and histone modification, often accompanying lack of *H3K27* trimethylation [147,149,150]. With the exception of some genomic imbalances, namely 1q gain and 6q loss, they typically demonstrate a balanced genome [139,147,149]. They are most common in infants and young children, have a tendency towards infiltration, dissemination and consequent poor prognosis [153]. Due to their predominant lateral location and inherent invasiveness, gross total resection (GTR) is often difficult to achieve and therefore relapse rates are high [154]. PF-B ependymomas are characteristically enriched with numerous cytogenetic abnormalities and are more common in adolescents and young adults [139,152,155]. They originate in the midline yet are often amenable to surgical resection, have a low metastatic potential and therefore have a superior outcome to PF-A tumors [139,152,155]. Recent methylation profiling work to further categories these two PF subgroups have reported two major subgroups, nine minor PF-A subtypes and five PF-B subgroups displaying variable clinical and genetic heterogeneity [140,156].

Greater than 70% ST ependymomas contain a zinc finger translocation associated (ZFTA, previously C11Orf95) gene fusion, most commonly RELA-ZFTA and are termed ST-RELA or, more recently ST-ZFTA [139,143,151]. This subtype is found in children and adults, but rarely infants and is often located in frontal or parietal lobes, often with intratumoral hemorrhage, cysts or necrosis [157]. ST-YAP is the remaining molecular subgroup, characterized by the fusion of the *YAP1* oncogene with *MAMLD1* [142]. ST-YAP tumors typically arise in ventricular or periventricular locations among infants [142]. Up to 15% of ST ependymoma may not harbor a *RELA* or *YAP1* fusion [158].

#### *4.4. Prognostic Factors*

Interest remains in identifying prognostic markers to aid patient risk stratification for future ependymoma trial design to improve upon the relative poor long-term outcomes that exist. Akin to medulloblastoma, several clinical and histological putative markers (location, age, tumor grade) have been rendered obsolete by the identification of molecular subgrouping.

The most consistent clinical marker is the extent of surgical resection, with some studies reporting a 60% difference in survival between cases of complete and incomplete tumor resection [59,120,132,159–162]. The positive prognostic effect of complete excision is maintained across molecular subgroups [120,139].

The infiltrative nature, localization and predisposition to metastasis suggests PF-A ependymomas should exhibit a poorer prognosis when compared with PF-B counterparts, an assumption supported by a retrospective analysis 820 patients with PF ependymoma across four independent cohorts [161]. The recent prospective Children's Oncology Group (COG) ACN0121 clinical trial, however, found no difference between PF-A and PF-B patient survival, although likely reflecting a paucity of PF-B cases [59]. The study did identify an adverse association with 1q gain in PF-A cases, with survival as low as 30% despite tumor resection and radiotherapy administration [59]. As stated above, tumor gain of chromosome 1q and loss of chromosome 6q are the most commonly observed chromosomal imbalances in ependymoma and appear adverse prognostic factors [59,120,139,141,144–146,148,152]. A recent retrospective molecular profiling study of 212 primary PF-B ependymomas identified loss of 13q as a potential novel adverse marker [140].

A retrospective cohort study of 122 ST ependymomas identified *ZFTA*/*RELA* fusion as a poor prognostic marker, regardless of the attainment of resection status, with 10 year PFS and overall survival (OS) of approximately 20% and 50%, respectively [139]. The same study conversely identified excellent ST-YAP1 survival rates of 100% [139]. Nevertheless, data from the ACNS0121 clinical trial failed to show any adverse prognostic implication for ST molecular subgroups, again potentially influenced by the case numbers involved [59].

#### *4.5. Current Management/Clinical Trials*

The globally accepted standard for pediatric IC ependymomas is maximal, safe surgical resection followed by involved field adjuvant radiotherapy (RT), dosed at 54–59.4 Gy, founded from a 2009 St Jude's Children's Research Hospital single-center study of 107 children, demonstrating a 7 year PFS of 77% and OS of 85% [160]. Exceptions to this are in metastatic cases where craniospinal radiotherapy is typically utilized for older children, and infant IC ependymomas, where a chemotherapy only strategy is reserved in order to avoid or delay radiotherapy to the developing brain, with eligibility thresholds of 12 to 18 months for PF tumors and up to 3 years for ST tumors.

Concerns regarding radiotherapy-induced neurotoxicity in young children have resulted in IC ependymoma being the most common pediatric tumor treated with proton beam radiotherapy. By reducing radiation exposure to healthy tissue while delivering therapeutic doses, this modality delivers comparable disease control to modern photon radiotherapy without unexpected toxicity [163–165]. Data continue to be collated on latent toxicity [164].

Recent, large international co-operative IC ependymoma trials have been designed to validate the findings of the 2009 St. Jude's study, evaluate the utility of an aggressive surgical approach, and verify a therapeutic role for chemotherapy either pre or post-radiotherapy (Table 1), since historical data have proven contradictory and inconclusive. The North American CCG-9924 study reported a PFS benefit from immediate post-operative chemotherapy prior to radiotherapy in patients where over 90% of the tumor has been resected [166]; however, this approach has been rebutted by other trial groups [167]. Similarly, outcomes from chemotherapeutic, radiation-sparing strategies for infants have been inconsistent and ultimately disappointing for the majority of children, with only a minority ultimately sparing radiation [168–171].

The COG ACNS0121 trial confirmed the efficacy of an aggressive surgical approach followed by immediate post-operative radiotherapy, even for children below 3 years of age when compared to historical controls [59]. Long-term follow-up of these younger patients is eagerly awaited. The impact of post-operative chemotherapy to facilitate secondlook surgery could not be determined. The COG ACNS0831 study followed on from ACNS0121, with the randomized addition of continuation chemotherapy (vincristine, cisplatin, cyclophosphamide and etoposide) for children treated with adjuvant focal RT following a complete or near total resection [60]. An interim "as treated" analysis of patients was undertaken due to significant non-compliance in patients randomized to receive chemotherapy. This reported a survival advantage for patients receiving chemotherapy (3 year EFS 80% vs. 71%; 1-sided *p*-value = 0.0121) [60].

The open phase II/III SIOP-Europe Ependymoma II trial (NCT02265770) has design similarities with the COG studies, making compliance with post-irradiation chemotherapy randomization imperative to validate the findings from ACNS 0831. Through patient allocation to three strata, the trial also attempts to evaluate the value of pre-radiotherapy chemotherapy and a 8 Gy radiotherapy boost in cases of incomplete resection, and the addition of a of a histone de-acetylase (HDAC) inhibitor, sodium valproate, for infants receiving one year of conventional multiagent chemotherapy.

#### *4.6. Novel Therapies*

Several biological models and patient derived xenografts have been developed to recapitulate ependymoma subgroups in order to identify new therapeutic targets and test novel therapies [172–174]. High throughput drug screening in murine models of *ZFTA* fusion-negative supratentorial ependymoma, characterized by the *Ephb2* oncogene identified 5-fluoracil (5-FU) as a potential active drug against this subtype [174,175]. Fibroblast growth factor receptor inhibitors have also been shown to have activity against patient derived ependymoma cell models and demonstrate efficacy in the clinic [176]. As detailed above, the use of histone deacetylase inhibitors as differentiation therapy is currently under evaluation in the current SIOP-Europe trial, following in vitro analyses [177,178]. Similarly, the phase I/Ib COZMOS trial is evaluating the DNA methyltransferase inhibitor 5 0Azacitidine in combination with carboplatin, on the premise that inhibition of aberrant DNA methylation will have therapeutic benefit (NCT03206021). Other novel therapies being explored include chimeric antigen receptor T-Cells (HER2; NCT03500991), based on encouraging pre-clinical murine work [80] and metronomic antiangiogenic therapy [179,180].

#### **5. Conclusions**

This review exposes the need for the pediatric neuro-oncology community to address the disparity that has developed between advances at the bench compared to the bedside. The potential for an era of biology driven patient care clearly exists yet, at present, international clinical trials struggle to keep pace with the scientific progress made to date. Indeed, many are being rendered outdated before they open to recruitment when evaluated against current molecular advances. This challenge is not unsurmountable and indeed should be embraced as recent years have demonstrated a paradigm shift in our understanding of the molecular pathogenesis across principal malignant brain tumor groups, therein serving as

the foundation for developing both risk stratification systems and novel agents as part of the next generation of clinical trials. Nevertheless, results from the review highlight that the statistical design, regulatory infrastructure and ultimately funding of such studies will need urgent consideration to achieve these objectives.

#### *5.1. Clinical Trials and Therapeutic Protocols*

We have shown that for pediatric medulloblastoma, the four established intrinsic molecular subgroups have now been superseded by the identification of up to 14 subtypes, each demonstrating a disparate corresponding clinical profile. In contrast, most treatment protocols over the past 20 years have continued to treat MBs with the historical backbone of craniospinal radiotherapy and multiagent chemotherapy, only recently tailoring therapy intensity according to WNT/non-WNT subgrouping, without particular focus on the three other subgroups. Encouragingly, open international trials are now attempting to stratify patients and adapt therapy according to molecular diversity. For example, the SIOP-Europe PNET5 study is following a risk-adapted treatment stratification according to low and high-risk WNT subgroups, the SHH-alpha MB subtype (which demonstrate *TP53* mutations), standard-risk biological profiles (including *MYCN* amplified Group 4 MB) and children with a germline mutational profile (NCT02066220). The SJMB12 trial, in addition to evaluating treatment de-escalation for WNT-subgroup patients, is assessing the addition of smoothened inhibitor Vismodegib for SHH MB, and the incorporation of gemcitabine and pemetrexed for high-risk Group 3 and 4 MB patients (NCT01878617). Finally, the SIOP-Europe high-risk medulloblastoma trial is using molecular screening to identify appropriate cases for increased-intensity treatments, including *MYC*/*MYCN* amplification (excluding *MYCN* amplified Group 4 MB) and SHH-alpha MB (EudraCT Number: 2018-004250-17).

Attempts to integrate molecular pathogenesis to inform on therapeutic stratification for most childhood high-grade gliomas or pediatric intracranial ependymoma unfortunately lag significantly behind the progress observed with medulloblastoma. As shown in this review, there is now compelling evidence that molecular subgrouping alone is an independent survival marker for childhood ependymoma, while prognostic adversity is further conferred by the presence of genomic aberrations including chromosome 1q gain and 6q loss in PF-A ependymomas, and potentially 13q loss in PF-B ependymomas. Despite this, international ependymoma clinical trials continue to risk stratify children according to the clinical parameters of patient age and resection status alone; an omission that will require addressing in future clinical trial strategies. With the exception of BIOMEDE 1, large-scale international pediatric HGG trials have also not incorporated biologically derived therapeutic stratification systems, principally because the finding that HGGs encompass an array of discrete subtypes is a relatively recent discovery.

As with medulloblastoma, the observation of up to 14 discrete molecular subtypes of PF ependymoma, at least 3 subtypes of ST ependymoma and up to 10 pediatric HGG subtypes clearly presents a challenge for future trial design. As can be seen from Table 1 of this article, the duration of an international pediatric brain tumor trial can take up to 10 years to complete patient accrual, and even longer to publish data. In order to tailor therapeutic intensity or introduce novel agents against the array of specific tumor subtypes now published in this review, future trials will require novel statistical designs that embrace truly global collaboration to generate timely, rigorous results as increasing molecular subcategorization will lead to significantly smaller patient subpopulations from which statistically sound conclusions must be drawn. Such collaborative efforts may also support less affluent countries to provide equity in diagnostic and therapeutic approaches. Duration of follow-up for specific patient populations will also need to be considered, as evidenced by the high proportion of late relapses in Group 3/4, subtype VIII MB and some non PF-A subgroups of ependymoma.

#### *5.2. Conventional and Novel Therapies*

While advances in adjuvant therapy have undoubtedly improved the survival of children with malignant brain tumors, the 'one-therapy-fits-all' paradigm fails to reflect and tailor to the diverse molecular landscape now apparent. As highlighted by this review, integrating clinical and biological data to generate risk-adapted treatment stratifications can potentially modify conventional therapy intensity and enable the introduction of novel agents.

De-escalation of radiotherapy dosing is being evaluated in several of the current international medulloblastoma clinical trials highlighted in the review. However, such an approach could also be considered for other molecularly-defined tumor entities including Group 4 (often subtype IV) medulloblastomas with chromosome 11 loss, completely resected ST-YAP1 ependymomas, completely resected PF-B ependymomas without 13q loss, and 'infant' or 'LGG-like' pediatric HGGs. Clearly, any de-escalation of therapy must be approached with extreme caution, as evidenced by the failure of trial NCT02212574 for WNT-activated MB, where a post-operative chemotherapy only strategy led to unacceptable relapse rates.

For some unfavorable-risk tumors, the option of increasing treatment intensity is a possibility as evidenced by current high-risk medulloblastoma trial strategies; however, any trial adopting this approach should consider incorporating disability or health status outcome measures, as they will help determine the quality of potential survivorship afforded [181]. The efficacy of chemotherapy in pediatric ependymoma remains contentious but a potential option to explore for escalation of therapy in certain cases (for instance PF-A tumors with chromosome 1q gain or 6q loss). The interim analysis results of the COG ACNS0831 trial suggested a potential survival advantage for children receiving continuation chemotherapy following tumor excision and post-operative irradiation, yet this requires validation ideally by the open phase II/III SIOP-Europe Ependymoma II trial. The administration of conventional chemotherapy agents and novel agents by alternative means, such as convection enhanced delivery to overcome the blood–brain barrier in diffuse midline glioma, H3K27M pediatric HGGs is also under consideration.

Parallel to modifying the intensity or administration of conventional therapies for childhood malignant brain tumors, much hope rests on establishing novel agents to target aberrant molecular aberrations underpinning tumorigenesis. This review highlights many of the developments in this field across medulloblastoma, pediatric high-grade gliomas and ependymomas. International trial outcomes are awaited for medulloblastoma subgrouptargeted therapy in SJMB12 and combination HDACi therapy across infants in the SIOP Ependymoma II study, while the success of BRAFi and NTRKi in certain pHGG subtypes and the evolving array of targeted primary treatment options for pediatric low-grade glioma give cause for optimism.

While encouraging, challenges nevertheless remain. As described in this review, novel agents against malignant brain tumors are being evaluated in early-phase pediatric studies, yet few successful candidates targeting the spectrum of molecular subtypes that now exist have been identified. One explanation for this is that many early-phase neuro-oncology trials in children assess novel agents in the relapse setting, rather than as primary therapy. In turn, this could potentially generate misleading results on drug efficacy, as evidenced by pre-clinical relapsed medulloblastoma work implicating clonal selection as a potential cause for the disappearance of targetable aberrations between patient-matched primary and relapsed tumors [182,183]. However, the paucity of effective novel agents also reflects the ongoing need for improved pre-clinical models that accurately replicate the specific human disease subtype interrogated, including appropriate immunocompetent murine models to test potential immunotherapies. A further explanation is that many pediatric malignant brain tumors appear driven by epigenetic dysregulation such that tumors rarely harbor immediately actionable mutations, or display significant molecular heterogeneity making resistance to single agent targeted therapy anticipated, as is described for SHH-activated medulloblastoma [30]. Consequently, it is presumed that combination therapy, utilizing

novel agents alongside conventional modalities, will better enable local and disseminated disease control rather than a single agent approach in future studies.

#### *5.3. Future Challenges*

This review highlights the molecular heterogeneity across the most common pediatric malignant brain tumors, together with its relevance to current diagnostic and therapeutic protocols, and strategies to correct the consequent imbalance that arises from bench to bedside. The tumor groups discussed in this review have key clinical challenges that now warrant focus, including intensification or novel combination therapy for unfavorablerisk tumors, de-escalation of intensity for favorable-risk lesions, the treatment of relapse, and a reduction in morbidity, disability and late effects (Table 2). It is now incumbent on the neuro-oncology community to meet and overcome these challenges; in an age of digital technology and social media, where the latest global scientific breakthroughs are acknowledged promptly in the public domain, the families of our patients are demanding this of us.

**Table 2.** Future clinical challenges for pediatric malignant brain tumors.

**Author Contributions:** Conceptualization, J.-P.K., C.T.; Writing-original draft preparation, J.-P.K., C.T.; Writing-review and editing, J.-P.K., C.T. All authors have read and agreed to the published version of the manuscript.

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

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

#### **References**


**2. Ayurveda**

Hippocrates, the father of modern western medicine, is famous for his expression "All disease begins in the gut." Ayurveda places great importance on proper diet and digestion, as well as on all aspects of lifestyle. Some researchers consider Ayurveda to be an ancient science of epigenetics [51]. The Ayurvedic practitioners might not have understood the precise nutrigenomic mechanics of how food can a ffects gene expression [52], but they did recognize that each individual has his or her own unique psychophysiological constitution, which is a ffected by diet, digestion, lifestyle, stress management, and environmental factors [53].

Ayurveda describes the functioning of our mind and body in terms of three main doshas—the governing principles of the physiology—Vata, Pitta, and Kapha [54]. In a previous paper, we have reviewed the research on the physiological, biochemical, and genetic correlates of each of the doshas. As we explain, the category of Vata dosha includes processes responsible for movement at all levels of the physiology, excretion of wastes, and also cognition. The primary location of Vata, according to

Ayurveda, is in the colon, where most of the gut microbiome is located. Pitta dosha is responsible for such functions as digestion, metabolism, thermoregulation, and energy homeostasis and resides in the mid gut area. Kapha dosha governs the growth and maintenance of structure and its primary location is in the chest area. Genetic research shows a significant difference between each dosha type and gene expression. Genes related to cell cycles were upregulated in the Vata types whereas genes in the immune response pathways were upregulated in the Pitta type, and genes in the immune signaling pathways were upregulated in Kapha types. Each dosha type is suggested to display its own style of brain functioning [55,56]. In Ayurveda, one of the first stages of an assessment of a person's health is to determine the distribution of the three doshas at birth, which is called Prakriti. In addition, an assessment is made of the state of balance of the doshas, which is called Vikriti. In fact, one can have a dominant Vata Prakrti at birth, but at the time of the diagnosis, the primary imbalance may be in Pitta due to some environmental factor.

In Figure 1, we illustrate that the microbiome is integral to both modern medicine and Ayurveda. We elaborate on this concept throughout the article.

**Figure 1.** Ayurvedic anatomy affected by the microbiome.

#### **3. Prakriti and Gut Bacteria**

One study has examined the relationship between Prakriti and the composition of the microbiome. The researchers found that three main Prakriti types, Vata, Pitta, or Kapha, had a unique microbiome composition [57]. The population studied were from the same region and had similar dietary habits. The main bacteria of all the subjects were from the phyla Bacteroidetes and Firmicutes. There were distinct differences between the Vata, Pitta, or Kapha groups in less common bacteria. The extreme Pitta individuals, for example, had more butyrate producing microbes which might help protect them from inflammatory diseases. The extreme Kapha women had larger amounts of a type of bacteria called *Prevotella copri*, which has been associated with patients who have rheumatoid arthritis and insulin resistance. A more recent paper analyzes how the concept of Prakriti can be used as a stratifier of the gut microbiome [58].

#### **4. Ayurveda Herbs and Spices and the Microbiome**

Ayurveda considers food as medicine. It uses spices and herbs to help create and maintain balance in the physiology, and to treat specific disorders. Recent research shows that both ginger and a herbal preparation called Triphala can have beneficial effects on the microbiome. Triphala consists of three fruits: Emblica officinalis (Amalaki), Terminalia bellerica (Bibhitaki), and Terminalia chebula (Haritaki). It is a fundamental component of Ayurvedic gastrointestinal and rejuvenation treatment programs, particularly to help improve elimination. Studies have found that Triphala

has different potential clinical applications which include appetite stimulation and a reduction in hyperacidity and constipation. It also has a number of inherent biological properties such as anti-inflammatory, immunomodulatory, antibacterial, antimutagenic, adaptogenic, hypoglycemic, antineoplastic, chemoprotective and radioprotective, and antioxidant activities. In the study, it was shown that the polyphenols in Triphala modulate the human gut microbiome and thereby promote the growth of beneficial Bifidobacteria and Lactobacillus while inhibiting the growth of undesirable gut microbes. The authors also suggest that the bioactivity of Triphala is elicited by gut microbiota that generate a variety of anti-inflammatory compounds [59].

Turmeric is another Ayurvedic preparation which has been found to affect digestion and the microbiome. Turmeric and its active ingredient curcumin have been the subject of thousands of peer-reviewed and published biomedical studies, with hundreds of potential preventive and therapeutic applications on such diseases as ulcerative colitis, stomach ulcers, osteoarthritis, heart disease, cancer, and neurodegenerative disorders [60,61]. Interestingly, the active ingredient of turmeric, curcumin, has been shown to be biological active but because of its poor bioavailability and inability to reach target tissues, it has not been found to be effective in clinical trials [62,63]. It may also be ineffective because we no longer have the synergic effects of the other ingredients. It is suggested that curcumin may restore dysbiosis of the gut microbiome and as a result have a neuroprotective effect, however, future research is needed to know how curcumin actually affects the microbiome in different individuals [64,65].

There are many other important Ayurveda herbs and preparations that need to be further studied. One of the most important categories are Rasayanas which are designed to lengthen the lifespan [66]. Once again, future research will be needed to examine the specific effects of Rasayana on the microbiome.

#### **5. Ama and Leaky Gut Syndrome**

Ayurveda explains that most diseases are caused by an accumulation of ama or undigested food. Ama literally means "uncooked food," but it can be understood from a scientific perspective as endogenous toxins resulting from imbalanced or incomplete digestion. Ama can be formed as a result of reduced Agni, or digestive power. Agni has a number of different meanings and not only relates to digestive enzymes but also to the metabolic process in the different tissues or dhatus of the body [67]. Ama is initially formed in the digestive tract, but at a later stage of disease it can leak into the bodily tissues and turn into Amavisha, a reactive form of ama, that leads to tissue disruption and chronic inflammation and disease.

It has been suggested that compromised mucosal integrity, such as a disruption in the tight junctions, leads to dysbiosis, resulting in the formation of Ama [68]. Ama is also produced at other levels of the physiology, including the cellular level. Excessive formation of free radicals contributes to the formation of Ama. A variety of free radicals and reactive oxygen species (ROS) are produced during cellular metabolism. Excessive amounts of these reactive molecules can cause damage, starting the disease process. The ability to control their concentrations may be helpful for the prevention and treatment of many disorders.

Antioxidants "scavenge" free radicals and ROS and render them harmless. Antioxidants can be lipid- or water-soluble; some are produced in the body and others are obtained from food or dietary supplements. Natural antioxidants range from vitamins to enzymes to herbal mixtures. Powerful antioxidants are present in the bioflavonoids found in concentrated form in Rasayanas. The use of these Rasayanas, might be helpful in neutralizing the excessive free radical activity that contributes to Ama formation. However, it is not clear if accumulated Ama in the body can be removed by the use of Rasayanas and antioxidants alone. Other Ayurvedic methodologies may be required to eradicate accumulated Ama. Rasayanas are best administered after a detoxification treatment such as panchakarma and are utilized to neutralize ongoing damage to the physiology and regenerate the system [68].

It is interesting to interpret the meaning of Ama in terms of recent findings. Remarkable progress has been made in understanding the mechanisms of leaky gut syndrome in celiac patients. In celiac disease, the tight junctions that hold the cells of the gut wall together become loose, and as a result, undigested food and harmful substances "leak" through the gut wall and into the bloodstream, causing inflammation. The exact mechanism of this process involves a product of gluten called gliadin, which interacts with receptors on the surface of the cells in the small intestine, causing the production of zonulin. Zonulin then causes the proteins, which bind tight junctions, to relax and allow unwanted substances to enter the bloodstream. In celiac patients, this process is exaggerated and can ultimately result in a harmful autoimmune reaction [69,70]. A disruption in the gut microbiome has been associated with problems in the gut barrier. It is suggested that gut dysbiosis may cause the release of zonulin but the exact mechanisms are complex and still under investigation [71,72].

#### **6. Biorhythms and Gut Bacteria**

Ayurveda clearly identifies daily, seasonal, and lifetime rhythms. Each day, for example, consists of a sequence of periods, which are characterized by Vata, Pitta, or Kapha. The day starts with a Kapha period from 6 am to 10 am, then a Pitta period from 10 am to 2 pm, and then a Vata period from 2 pm to 6 pm. Next is another Kapha period from 6 pm to 10 pm, a Pitta from 10 pm to 2 am and finally a Vata from 2 am to 6 am. Each season is represented by either one dosha or a combination of doshas: late autumn and winter are cold and dry and correspond to the Vata dosha. Summer is hot and naturally corresponds to Pitta dosha. Spring is cold and wet and corresponds to Kapha dosha. The daily routine is called dinacharya and the seasonal routine is called ritucharya.

Modern medicine also recognizes the importance of daily rhythms to your health and the 2017 Nobel Prize in Physiology and Medicine was awarded for research on the genetic basis of biological rhythms. From bacteria to humans, almost all forms of life have an internal "biological clock," which maintains an approximately 24 h rhythm. When external signals of light and dark are introduced unnaturally, the master clock becomes confused and this creates health problems. Shift workers, for instance, have been shown to have a higher incidence of cancer, cardiovascular disease, digestive disorders, and obesity, as well as psychiatric and neurodegenerative diseases [73].

One of the most important timing issues for the body, which has been seen in research on animals and humans, is when you eat. If you eat within 2 h before you normally go to sleep, it can desynchronize the circadian rhythms of certain cells in the intestine and liver from those in the rest of your body [74].

Gut bacteria also have biological rhythms. One study created jet lag in mice by forcing these normally nocturnal animals to stay awake during the day. When the researchers transferred the gut bacteria from jet-lagged mice into germ-free mice, the recipient mice developed both obesity and glucose intolerance [75]. A number of studies have shown that time-restricted feeding and eating frequency affects the composition of the microbiome [76]. Recent research on the gut bacteria reveals not only the presence of a daily rhythm, but also seasonal biorhythms [77,78]. The relationship between the circadian rhythms and gut microbiota appears to be bidirectional and may have important influences on health [79–82].

Ayurvedic recommendations of specific diets at specific times may relate to the seasonal rhythms of the microbiome. Over the winter, for example, Kapha and Ama are said to build up in the body. Early spring is described as the ideal time to rebalance Kapha and reduce Ama to prevent toxins and excess mucus from creating congestion and allergies. Foods that are primarily Kapha in nature—heavy, greasy, and mucus forming—tend to increase both Kapha and Ama. Ayurveda recommends reducing or eliminating Kapha foods during this time, which might help to heal the gut and reboot the microbiome [83].

#### **7. Probiotic Enemas and Bastis**

Probiotic enemas have been suggested to help cure certain neurological disorders [84]. Probiotics that are taken orally must pass through your stomach and the small intestine, which is not ideal because stomach acid and digestive enzymes destroy many of the valuable probiotic bacteria long before they reach the colon. A probiotic enema, however, provides an almost instantaneous route to the colon, where most of the gut bacteria live.

The Ayurvedic term for enema is basti, and bastis are a valuable part of the deep purification and detox treatment program known as panchakarma, which, as we mentioned, cleanses the body of impurities and promotes health and longevity. There are many types of bastis: some are for purification, others for elimination, others strengthen the tissues and provide valuable nutrients. Bastis use ingredients such as sesame oil, medicated ghee, buttermilk, lassi, many di fferent combinations of herbs, and in special cases, bone broth. A search of Pub Med shows a number of published studies on di fferent types of bastis that have been used for various clinical applications [85–92].

These studies, however, do not mention the use of bastis as a means to modify or reboot the gut bacteria. By studying how bastis a ffect gut bacteria composition we might gain a better scientific understanding of the mechanisms of their beneficial actions on specific health conditions. We already know that sesame oil has a positive e ffect on colon cells, and on certain beneficial bacteria [93,94].

In a more recent study, the metabolomic profile was taken after a 6-day panchakarma treatment. The experimental group consisted of healthy male and female subjects whose treatment program included herbs, vegetarian diet, meditation, yoga, and massage. The results showed that 12 plasma phosphatidylcholines decreased after treatment in the experimental group as compared to the control group of 54 subjects. There were changes in metabolites across many pathways such as phospholipid biosynthesis, choline metabolism, and lipoprotein metabolism, with statistically significant changes in the plasma levels of phosphatidylcholines, sphingomyelins and others in just 6 days. It is unclear whether the lipid metabolites were modulated by the gut microbiome or by the external agents such as herbs, vegetarian diet, meditation, yoga, and massage [95].

The study of panchakarma and other Ayurvedic purification treatments is an important area of future research. It would also be useful to compare the modern use of fecal transplant treatment with all the di fferent types of basti treatments.

#### **8. The Gut–Brain Axis and Ojas**

The gut–brain axis, as we described earlier, consists of a number of major systems in the body, as well as the gut bacteria. The brain communicates to the gut through either nerves or through hormones and chemical messengers. The gut–brain axis includes the enteric nervous system (ENS), enteroendocrine system (EEC) and the gut bacteria, which all produce di fferent chemicals. The ENS produces more than 30 neurotransmitters. Almost 90% of all the serotonin in the body is produced by cells in the gut, as well as 50% of the dopamine [39–41,96].

According to Ayurveda, the digestion and gut play leading roles in immunity. Ayurveda speaks about a substance or a process called Ojas, the finest product of healthy digestion, which strengthens the immune system and has many beneficial e ffects on the mind and body.

Can Ojas be identified by modern science? In Ayurveda there is a description of seven basic tissues or dhatus and three waste products or malas. The tissues roughly correspond to the tissues as discussed in modern physiology and the waste products include feces, urine, and sweat. The tissues are important because digestion includes the transformation of food into each of these tissues and ultimately to Ojas. It is unclear what this means in terms of modern science. It might correspond to a metabolic process that involves specific precursors which come from a well-functioning digestive system and a balance microbiome.

There are several possible candidates for Ojas. One is serotonin, a key regulator of mood, sleep, appetite, and other brain functions. As we mentioned, the gut produces most of the serotonin in the body, which circulates throughout your bloodstream and influences not only your immune system, but your heart rate, blood clotting, intestinal motility, pulmonary arteries, heart, brain, and mammary glands, as well as the cell growth of liver and bone cells [97]. One of the key precursors of serotonin is tryptophan, an essential amino acid, which is obtained through the normal digestion of certain foods.

Ojas is described as being beneficial to all parts of the body so we might ask if excess serotonin has any deleterious effects. An overabundance of serotonin due to drugs can lead to a number of damaging symptoms, including anxiety, sweating, sleep disturbance, appetite changes, headaches, confusion and lethargy, dilated pupils, rapid heart rate, etc. [98]. Additionally, individuals with social phobia have been shown to have excess serotonin [99].

Another interesting candidate is a chemical called butyric acid, which, as we mentioned, is a by-product of the gut bacteria and has numerous beneficial effects, including improvement of immunity [100].

Ojas is described in the Ayurvedic texts as having many properties, such as its ability to coordinate the junction between the mind and body. Some of these effects may not be covered by either serotonin or butyric acid. The exact nature of Ojas remains an ongoing research project.

#### **9. Stress, Ayurveda, and Psychobiotics**

Stress can have many deleterious effects. When the brain triggers the adrenal glands to release cortisol, it goes into the bloodstream and affects both the gut and the gut bacteria. Cortisol can increase the intestinal permeability, which results in a leaky gut. It can also shut down the activity of the gut immune system [71,101].

Ayurveda uses a wide variety of approaches to improve the mind or manas and to alleviate the effects of stress. One of these is meditation, which is an integral part of both Ayurveda and its sister discipline, yoga. The Transcendental Meditation (TM) program introduced by Maharishi Mahesh Yogi over fifty years ago has been shown to have many distinct physiological changes, including a reduction in cortisol, as well as beneficial effects on mental and physical health [102–107]. To date, we know of no studies which have examined the effect of meditation on the gut microbiome.

Ayurveda has long explained that disturbances in our mental state, which are usually associated with a Vata imbalance, can be traced to problems in the nervous system and the gut. It recommends specific herbs such as Ashwagandha and Brahmi, which may interact with the microbiome. It also has a long tradition of using natural probiotics such as lassi. Modern research on the microbiome validates this concept, showing that depression and anxiety may be linked to a disrupted microbiome and may be improved through the use of probiotics.

One brain imaging study showed that women react differently to stimuli depending on the type of bacteria they have in their gut. The same researchers had previously shown that diet could affect the brain. They gave subjects either a psychobiotic (a probiotic mixture that is used for mental health) or a placebo.  The subjects taking the placebo showed a normal stress response with specific areas of the brain responsible for emotions being activated. Subjects receiving the psychobiotic showed a reduced stress response in these same areas of the brain [108,109].

#### **10. The Future of Ayurveda and Modern Medicine**

We are living in a transitional moment in medicine. New studies are emerging that will help us make better choices. The field of integrative medicine represents an important achievement since it combines the best of modern medicine with the best of traditional system of natural health, such as Ayurveda. Integrative medicine benefits greatly from research on the microbiome because it helps us better understand the ancient practices of Ayurveda in the light of modern science [110].

**Funding:** This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. A grant from the Wege Foundation helped pay for publication costs.

**Acknowledgments:** Thanks to Roxanne Medeiros for creating the graphic.

**Conflicts of Interest:** The author declares no conflict of interest.