*Review* **The Psychoneuroimmunology of Stress Regulation in Pediatric Cancer Patients**

**Gillian E. White <sup>1</sup> , Jessica E. Caterini <sup>1</sup> , Victoria McCann <sup>2</sup> , Kate Rendall <sup>1</sup> , Paul C. Nathan <sup>3</sup> , Shawn G. Rhind 4,5 , Heather Jones <sup>3</sup> and Greg D. Wells 1,\***


**Simple Summary:** There are many commonalities between children with cancer and other populations that experience early-life stress. Thus, it is important to review the existing research surrounding the stress response in the pediatric cancer population. In this review, we describe the psychoneuroimmunology behind stress regulation and the differences observed in stress regulatory pathways in childhood cancer patients. Our objective is to provide a clinically relevant summary of the stress pathways contributing to, and exacerbating, childhood illness and outline some potential interventions.

**Abstract:** Stress is a ubiquitous experience that can be adaptive or maladaptive. Physiological stress regulation, or allostasis, can be disrupted at any point along the regulatory pathway resulting in adverse effects for the individual. Children with cancer exhibit significant changes to these pathways in line with stress dysregulation and long-term effects similar to those observed in other early-life stress populations, which are thought to be, in part, a result of cytotoxic cancer treatments. Children with cancer may have disruption to several steps in the stress-regulatory pathway including cognitiveaffective function, neurological disruption to stress regulatory brain regions, altered adrenal and endocrine function, and disrupted tissue integrity, as well as lower engagement in positive coping behaviours such as physical activity and pro-social habits. To date, there has been minimal study of stress reactivity patterns in childhood illness populations. Nor has the role of stress regulation in long-term health and function been elucidated. We conclude that consideration of stress regulation in childhood cancer may be crucial in understanding and treating the disease.

**Keywords:** stress; allostasis; stress regulatory pathways; stress dysregulation; stress reactivity; early life stress; childhood cancer; cytotoxic cancer treatment; interventions

### **1. Introduction**

Stress is a ubiquitous experience with significant impacts on health and function. Stress refers to the physiological state of the body in response to a stressor, whereas the stressors themselves are challenges, threats, demands and constraints that provide barriers to the normal daily functioning of the individual, thereby inducing stress and its related consequences on the body [1]. Stress responses function acutely to defend homeostasis during changing internal and external demands [2,3] and transduce external stimuli into physiological signals to support effective navigation of the environment and encode relevant information for future events [4]. Depending on the nature of the stressor and the

**Citation:** White, G.E.; Caterini, J.E.; McCann, V.; Rendall, K.; Nathan, P.C.; Rhind, S.G.; Jones, H.; Wells, G.D. The Psychoneuroimmunology of Stress Regulation in Pediatric Cancer Patients. *Cancers* **2021**, *13*, 4684. https://doi.org/10.3390/ cancers13184684

Academic Editors: Saurabh Agarwal and Jianhua Yang

Received: 30 July 2021 Accepted: 17 September 2021 Published: 18 September 2021

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**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/).

interpretation of it, stress can be adaptive ("eustress"), broadening one's ability to cope with environmental conditions and challenging experiences effectively, or maladaptive ("distress"), straining one's ability to cope and contributing to general dysfunction [2,5,6].

Significant or chronic exposure to stress activation can cause long-term changes to how the stress-regulatory system responds to future stressors [6–8]. This is particularly true of significant stress exposure during times of developmental plasticity, such as childhood and adolescence [9–13]. Several chronic diseases in adults and children, including cardiovascular disease, metabolic conditions, cancer and immunologic conditions have been attributed to chronic stress and/or dysfunctional stress regulation, in addition to a number of psychological and cognitive conditions and nonclinical functional outcomes following significant or chronic stress exposure [5,14–16]. Risk and prevalence of these stress-related adverse outcomes have been reported in populations of children experiencing significant stress or trauma [17] and are thought to be a result of alterations in stress regulatory pathways which can be detected by examining patterns of physiological signalling in response to an acute stress exposure [5,14–16].

While the adverse outcomes of childhood trauma and chronic stress have been well documented, current definitions of Adverse Childhood Events (ACEs) and early life stress models from which this research draws [4,9,10,13,15,17,18] do not include childhood illness. Stress dysregulation may provide a valuable paradigm through which to understand long-term health and wellbeing in populations experiencing childhood illness, given psychological strain and physically aggressive medical treatments.

Approximately 1 in 300 children will develop cancer between birth and 20 years of age [19] which involves significant psychological burden, physical distress and barriers to engaging in positive stress coping behaviour such as pro-social and physical activity habits. Children with cancer and survivors show evidence of neurological [20–27], physiological [28–34] and psychosocial [24,25,27,35,36] changes potentially attributable to stress dysregulation (see Figure 1). Further, they report higher risk of late effects similar to those reported in other early life stress populations, suggesting that stress dysregulation may be worth investigating as a putative pathway, or as a modulator of the damage caused by cancer treatment. Psychoneuroimmunology, a field of study that is inherently implicated in stress regulation, examines the relationships between human behaviour, the nervous and endocrine systems, and the immune system [37]. This review describes the psychoneuroimmunology of stress regulation and support for potential clinical relevance in childhood cancer patients and survivors [38–40].

**Figure 1.** Illustration representation of the interrelationships between childhood cancer and its treatment and the physical, neurological and psychosocial effects that lead to chronic stress. A bidirectional relationship exists between physical, neurological and psychosocial effects, as well as between chronic stress and dysfunction in these systems. The major health related effects of chronic stress leading to disease risk and lower quality of life. **Figure 1.** Illustration representation of the interrelationships between childhood cancer and its treatment and the physical, neurological and psychosocial effects that lead to chronic stress. A bidirectional relationship exists between physical, neurological and psychosocial effects, as well as between chronic stress and dysfunction in these systems. The major health related effects of chronic stress leading to disease risk and lower quality of life.

#### **2. Stress Regulation & Allostasis 2. Stress Regulation & Allostasis**

Selye defines stress as the body's adaptive response to a "noxious agent", involving a deviation from resting state, or homeostasis [2]. Homeostasis refers to the maintenance physiological variables essential for human life, such as internal body temperature, or pH, within a precise range [3]. Physiological stress regulation, also termed 'allostasis', is an adaptive process aimed at keeping the body's systems in physiological ranges, or maintaining homeostasis, despite changing internal and external environmental conditions [3]. Stressors can be both biological or psychological, and thus can be real or perceived, past, current, anticipated or recalled [41]. Since biological stressors are typically internal, they evoke a direct physiological response without the engagement of higher order cognitive processing [42]. Psychological stimuli, however, must undergo interpretation, and thus elicits a physiological stress response indirectly [14,43–45]. Therefore, a psychological response is mounted in response to the perception of stress, rather than the stressor itself. For the purposes of this review, we will only consider psychological stressors and the allostatic processes that they evoke. Selye defines stress as the body's adaptive response to a "noxious agent", involving a deviation from resting state, or homeostasis [2]. Homeostasis refers to the maintenance physiological variables essential for human life, such as internal body temperature, or pH, within a precise range [3]. Physiological stress regulation, also termed 'allostasis', is an adaptive process aimed at keeping the body's systems in physiological ranges, or maintaining homeostasis, despite changing internal and external environmental conditions [3]. Stressors can be both biological or psychological, and thus can be real or perceived, past, current, anticipated or recalled [41]. Since biological stressors are typically internal, they evoke a direct physiological response without the engagement of higher order cognitive processing [42]. Psychological stimuli, however, must undergo interpretation, and thus elicits a physiological stress response indirectly [14,43–45]. Therefore, a psychological response is mounted in response to the perception of stress, rather than the stressor itself. For the purposes of this review, we will only consider psychological stressors and the allostatic processes that they evoke.

The allostatic processes are an integration of subjective, often subconscious appraisal of a physical or psychological stimulus, which is converted into neural and endocrine signalling in turn activating target organs that elicit an allostatic response as shown in Figure 2. Effective allostatic responses are characterised by rapid upregulation of signaling hormones and effector tissue activation, commensurate with the level of threat encountered, and rapid termination upon cessation of stimulus—producing maximal physiological response to effectively defend homeostasis during stress experience with minimal exposure to signalling biomediators and associated energy costs [46]. The allostatic processes are an integration of subjective, often subconscious appraisal of a physical or psychological stimulus, which is converted into neural and endocrine signalling in turn activating target organs that elicit an allostatic response as shown in Figure 2. Effective allostatic responses are characterised by rapid upregulation of signaling hormones and effector tissue activation, commensurate with the level of threat encountered, and rapid termination upon cessation of stimulus—producing maximal physiological response to effectively defend homeostasis during stress experience with minimal exposure to signalling biomediators and associated energy costs [46].

ostasis.

**Figure 2.** A systems approach to stress responses. Some stimulus—internal or external, perceived or real, past, present or future—is interpreted as threatening through cognitive appraisal and integrated with affective feedback. Together, the cognitive-affective appraisal determines the saliency of the threat. If sufficient, this will cause neurological triggering of stress activation pathways in the brain, resulting in increased biochemical signaling of these pathways and their subsequent physiological activation of target organs. If a stable state cannot be achieved or maintained to meet the demands of the stressor, then excessive activation and associated wear on target tissues may result and possibly lead to dysfunctional signalling with excessive feedback by mediators on earlier pathway steps. Adapted with permission from ref. [14]. Copyright 2012 Springer. **Figure 2.** A systems approach to stress responses. Some stimulus—internal or external, perceived or real, past, present or future—is interpreted as threatening through cognitive appraisal and integrated with affective feedback. Together, the cognitive-affective appraisal determines the saliency of the threat. If sufficient, this will cause neurological triggering of stress activation pathways in the brain, resulting in increased biochemical signaling of these pathways and their subsequent physiological activation of target organs. If a stable state cannot be achieved or maintained to meet the demands of the stressor, then excessive activation and associated wear on target tissues may result and possibly lead to dysfunctional signalling with excessive feedback by mediators on earlier pathway steps. Adapted with permission from ref. [14]. Copyright 2012 Springer.

Allostasis is considered to be a mechanism by which the body can maintain homeostasis in the face of variable environments—stability through change [47]. To this end it can be considered from an energy conservation perspective in which the physiological responses are predictive and aim to maximise energy efficiency in coping with environmental demands [48]. It can also be considered a calibrating mechanism in which the mediators of the response feedback to the earlier steps to adapt optimally to environmental contexts [4]. The hypothesis of allostasis as a mechanism in long-term health and functional outcomes in childhood cancer patients and survivors is rooted in the data indicating altered regulation of the cognitive, psychological, physiological pathways that regulate physiological responses to stress. We suggest that understanding and mitigating this dysregulation of physiological responses to stress is integral to improving care in this population. Therefore, for the purposes of this paper, we take the view of allostasis as the process by which the body responds physiologically to stressors in order to regain home-Allostasis is considered to be a mechanism by which the body can maintain homeostasis in the face of variable environments—stability through change [47]. To this end it can be considered from an energy conservation perspective in which the physiological responses are predictive and aim to maximise energy efficiency in coping with environmental demands [48]. It can also be considered a calibrating mechanism in which the mediators of the response feedback to the earlier steps to adapt optimally to environmental contexts [4]. The hypothesis of allostasis as a mechanism in long-term health and functional outcomes in childhood cancer patients and survivors is rooted in the data indicating altered regulation of the cognitive, psychological, physiological pathways that regulate physiological responses to stress. We suggest that understanding and mitigating this dysregulation of physiological responses to stress is integral to improving care in this population. Therefore, for the purposes of this paper, we take the view of allostasis as the process by which the body responds physiologically to stressors in order to regain homeostasis.

Acutely, allostasis is adaptive and functions to maintain homeostasis in face of a prospective disruption by mediating a fight or flight response [8]. Biomediators of allostasis, specifically cortisol, also provide feedback to brain regions responsible for triggering allostatic responses in a self-regulating process that tunes the response to the stressor presented and terminates the acute activation of allostasis [6–8]. This same biochemical feed-Acutely, allostasis is adaptive and functions to maintain homeostasis in face of a prospective disruption by mediating a fight or flight response [8]. Biomediators of allostasis, specifically cortisol, also provide feedback to brain regions responsible for triggering allostatic responses in a self-regulating process that tunes the response to the stressor presented and terminates the acute activation of allostasis [6–8]. This same biochemical feedback calibrates the system to future stress exposures through both neurological encoding and affective working memory [4,6,8].

back calibrates the system to future stress exposures through both neurological encoding and affective working memory [4,6,8]. There are important functional consequences of chronic allostatic stress. The catabolic and energy costly processes needed to mount allostatic responses contribute to wear and tear on target organs, potentially exacerbating underlying weakness or dysfunction in these tissues. Further, the chronic feedback of biomediators on stress regulatory corticolimbic brain centres, particularly the hippocampus, alter their structure and function, ultimately impacting the overall regulation and responsivity of the system [49–54]. Due to the self-regulatory nature of stress regulation, dysregulation can occur with disruption to any point in the pathway shown in Figure 2. Typically, dysregulation that contributes to stress-related health and dysfunction is characterised by one of four patterns of stress There are important functional consequences of chronic allostatic stress. The catabolic and energy costly processes needed to mount allostatic responses contribute to wear and tear on target organs, potentially exacerbating underlying weakness or dysfunction in these tissues. Further, the chronic feedback of biomediators on stress regulatory corticolimbic brain centres, particularly the hippocampus, alter their structure and function, ultimately impacting the overall regulation and responsivity of the system [49–54]. Due to the selfregulatory nature of stress regulation, dysregulation can occur with disruption to any point in the pathway shown in Figure 2. Typically, dysregulation that contributes to stressrelated health and dysfunction is characterised by one of four patterns of stress reactivity: (1). Overly frequent activation; (2). No habituation to familiar stimuli; (3). Inefficient termination; or (4). Hypoactivation of one pathway resulting in compensatory overactivation of the other [3].

While stress can contribute to the development or acceleration of illness, it does not cause illness or dysfunction per se. Rather, the adaptive function of allostasis requires systemic upregulation. Often these are catabolic processes, resulting in wear and tear over time or "allostatic load" [3,10,18,55,56], which describes the cost that this prolonged upregulation and activation of compensatory effectors has on the body [57]. Allostatic load exacerbates existing tissue weaknesses, whether hereditary genetic predispositions or susceptibility from a previous insult, or a concomitant one such as cancer therapy. The aggregate of this subclinical dysfunction can have significant health and functional implications and has been associated with all-cause morbidity and mortality [3,55].

Much research to date has focused on individuals who experienced significant childhood adversity or ACE's (such as physical or emotional child abuse, neglect, parental substance abuse, household and family turmoil, etc.) but leaves out childhood illness [4,7,10,11,17,18,58]. Many commonalities exist between populations experiencing psychosocial early life stress and children experiencing childhood illness [13,17,18,28,58–60]. This presents the possibility that children treated for illness many be susceptible to long-term health and dysfunction associated with altered stress regulation. Childhood cancer patients experience significant stress prolonged over the duration of treatment, as well as significant physical and psychosocial effects of treatment and disruptions to their normal developmental opportunities [23,61]. As a disruption to any step in the pathway can result in dysfunctional regulation, we will review the role of each step and evidence of prospective dysfunction in childhood cancer patients and survivors.

#### **3. Cognitive-Affective Appraisal**

Allostatic processes are regulated by the integration of inputs to corticolimbic brain regions including the prefrontal cortex (PFC), hippocampus, amygdala and brainstem. These regions are implicated in many other functions besides stress circuitry such as decision making and higher cognition, learning and memory, emotional processing and the judgement of salience, among others [62]. Neurocognitive and neuropsychological function development corresponds with the timing of brain development through childhood and adolescence. In brief, functions associated with emotional reactivity develop ahead of those necessary for cognitive reappraisal and self-regulation during childhood [12,63], in such a way that the time-lag in development in conjunction with social contexts of adolescence is often attributed to the behavioural trends (impulsivity, risk taking) and greater stress reactivity seen during adolescence [12,64].

For evolutionary reasons, stimuli that involve social judgement, are goal oriented and include unpredictability, novelty and uncertainty are most salient and reliably evoke a physiological response [65,66]. The subjective nature of cognitive-affective appraisal presents significant inter-individual variability and is influenced by lived experience, disposition, cognitive and psychological strategies, as well as positive coping behaviours such as physical activity and social connections. Human and animal models of chronic stress have shown impairments to memory and executive functions, and these also appear to be long-term deficits reported in children and adults who have experienced early life stress [20,49].

Studies investigating the neurocognitive consequences of chemotherapy during childhood (see Figure 1) have also found impairments to working memory and executive function [20,67–69], as well as processing speed, task efficiency, attention, memory and learning [70–72]. An important association has been made between these functions and self-regulatory behaviour, such as effective coping [73]. As noted by Campbell et al. (2007), the consequences of these stress-induced impairments are present not only in school settings, but in many other domains of life such as social relationships, emotional control, coping skills, the workplace and overall quality of life [70]. In line with this, Krull et al. (2013) found that over 10 years post-treatment, 28–59% of childhood cancer survivors

reported neurocognitive and neuropsychological impairments, the greatest being reduced attention and executive functions, which were most closely associated with treatment with dexamethasone, a synthetic glucocorticoid, when excluding those who received cranial radiation therapy [35]. They further found that survivors reported an approximately 5% annual increase in self-reported behavioural issues related to self-regulation, which impacted functioning in academic and occupational settings [35].

Additional risk to effective cognitive-affective stress appraisal may be conferred by lower engagement in stress buffering behaviours such as socialisation and physical activity that promote adaptive coping [74–76]. Indeed, in a recent study of childhood Acute Lymphoblastic Leukemia (ALL) survivors, correlations were found between low levels of physical activity and inattention [77]. Further, disruption to corticolimbic brain regions central to these interpretive processes has been reported in several childhood cancer populations and is thought to be a result of cytotoxic treatments including chemotherapies that are neurotoxic and/or cross the blood brain barrier.

#### **4. Neurological Triggering**

During threat appraisal, corticotrophin-releasing hormone (CRH) is released from the hypothalamus and activates the hypothalamic-pituitary-adrenal (HPA) axis, while norepinephrine from the Locus-Coeruleus activates the autonomic nervous system (ANS), and ANS input directly activates target organs, and the sympatho-adreno-medullary (SAM) pathway [78]. The glucocorticoid cascade hypothesis posits that significant or enduring stress exposure will result in excessive cortisol exposure leading to altered hippocampal functional control over HPA scaling and termination, which in turn leads to further cortisol exposure and propagation of dysregulated stress signalling, ultimately leading to adverse health and functional effects [79]. Critically, the biomediators released by neurological triggering (cortisol and catecholamines) feed back to the brain, influencing both the cognitive-affective experience of stress, as well as continued neurological triggering. Cortisol feedback in particular is thought to be critical to tuning and terminating the activation of the HPA axis, by influencing neurotransmission of the hippocampus. Cortisol-mediated neuroplastic and neurotransmission changes to the hippocampus are commonly thought to be integral to the development of stress dysregulation. This may be of particular interest to populations treated with pharmacological glucocorticoids, such as prednisone and dexamethasone, both of which have CNS penetrance and are used for CNS prophylaxis for pediatric leukemia patients [23].

The protracted nature of brain development is such that subcortical and limbic structures (amygdala, hippocampus, brain stem) development precedes prefrontal and frontal cortex development [63,80,81]. Even though most adult hippocampal networks are apparent during childhood, their connections to lateral lobes increase throughout childhood (ages 4–10) [81] and PFC development is not fully developed until early adulthood [82].

Significant evidence in both human and animal models have found changes to neurological structures responsible for cognitive-affective processing and neurological triggering to be affected by exposure to chronic stress or early life stress [18,49,83–88]. Chronic stress in animal models has shown reduced hippocampal volume [83], reduced neurogenesis in the hippocampus [84–86], PFC atrophy [49] and amygdalar hypertrophy [49], concomitant with altered cognitive functions associated with those regions [49,83–86]. Human studies have found similar changes in altered hippocampal structure and function [18,87], PFC impairment [18,88] and other neurological impairments [88] in populations experiencing early life stress. Some inconsistencies exist in neurological studies of children, which have been attributed to the protracted nature of neurological development, such that the full extent of impact of early life stress on regional brain structure and function does not become apparent until the third decade of life [12,81,89]. It follows that the nature of brain changes is also sensitive developmentally, such that the regions that are in development when the stress occurs are most likely to experience long-term dysfunction [61,90].

Disruption to corticolimbic brain regions critical to stress regulation have been reported in childhood cancer patients treated with chemotherapy and/or cranial radiation. It is important to note that most studies examining the relationship between chemotherapy and brain structure and function changes have been performed mostly using cross-sectional study designs, as detailed in Table 1. Many of the neurological consequences associated with chemotherapy, including lower white matter volume [22,91–93], altered hippocampal microstructure [84,86,94] and altered PFC microstructure [73], are likely to impact neurological triggering and feedback effects on cognitive-affective appraisal. Both human [67,94] and animal [84–86] studies have reported lower hippocampal volumes and impaired neurogenesis related to various chemotherapeutic agents, similar to findings in other early life stress studies [18] (see Table 1). Amygdala changes have also been reported in adult cancer populations [26,94,95] and have been related to adverse psychological effects [26,95], and recently, reduced amygdala and dorsal striatum brain matter volume has been found in pediatric cancer populations [96]. Chemotherapy-treated survivors of childhood cancer displayed lower cerebellar volumes, versus healthy controls, which was associated with both poorer performance on neurocognitive testing and exposure to dexamethasone [97]. Despite some evidence of altered limbic and subcortical structure and function following chemotherapy, the majority of studies report differences in frontal and pre-frontal brain structures, as well as impairments in the function of these structures [20,21,35,67,70,98–100]. This may be due to the importance of these regions in global intellect and other neurocognitive deficits that have taken priority in this research space. The functional implications of these brain changes have not been studied in the context of stress, however, adrenal insufficiency and HPA dysfunction has been reported in children receiving cranial radiation suggesting that disruption to corticolimbic brain regions can have downstream effects on physiological stress signalling.









Abbreviations: MRI, magnetic resonance imaging; MB, medulloblastoma; CNS, central nervous system; ALL, acute lymphoblastic leukemia; MTX, methotrexate; DX, dexamethasone; AML, acute myelogenous leukemia; fMRI, functional magnetic resonance imaging; BOLD, blood-oxygen-level-dependent; DNA, Deoxyribonucleic Acid; TAC, total antioxidant capacity; TOS, total oxidant status; OSI, oxidative stress index; CRP, c-reactive protein; EAA, epigenetic age acceleration. Table 1 inclusion criteria: A scoping review of the literature was completed examining: (1). Primary research articles examining structural and functional neurological impairments in pediatric cancer patients; (2). Primary research articles examining inflammatory consequences and biological or cellular aging in pediatric cancer patients; (3). Pediatric cancer patients or survivors of childhood cancer (pediatric and adult).

> Whether these neurological changes are a direct cause of anti-neoplastic treatment (i.e., glucocorticoids, intrathecal methotrexate, cranial radiation) or endogenous stress processes, all can disrupt effective stress regulation and therefore, may be relevant to supporting long-term health and function in childhood cancer patients and survivors.

#### **5. Physiological Stress Response**

The signalling response evoked by neurological triggering is comprised of three distinct but overlapping systems. Brainstem activation of the ANS occurs almost immediately via neural inputs to visceral target organs activated for allostasis as well as through adrenal stimulation of catecholamines into circulation (SAM), while the hypothalamus activates the HPA axis via stimulation of the pituitary to release ACTH. These two arms of allostatic control, SAM signalling via catecholamines and HPA signalling via glucocorticoids, are responsible for the physiological and affective experience of stress.

Secondary signalling by inflammatory factors is also implicated in short and long-term effects and experiences of stress. The biomediators of inflammation, cytokines, can be produced by neutrophil demargination and activation of immune cells [109]. In the short term, this primes the immune system to protect the body from impending injury but in the long-term can contribute to reduced immune function, worse tissue healing and chronic inflammation and associated physiological and psychological disturbance [79,110].

Basal HPA and HPA-reactivity increases with age, with a marked increase around puberty (—13 for girls, 15 for boys) [64,111,112]. This is likely due to the effects of changing environmental demands, developing neurological structures that enable relevant neurocognitive and neuropsychological functions (i.e., goal-oriented behaviour is governed by the PFC and is thought to be an essential component of psychological stress provocation), and hormonal changes associated with puberty [88,111,113].Stress response patterns can be used to infer how the whole system is functioning and whether an individual appears

to have a resilient or vulnerable stress phenotype. These acute patterns are thought to be demonstrative of longitudinal stress regulation and prospective allostatic load and associated consequences for health and wellbeing. In a resilient and optimised system, the response pattern should show an immediate increase with stressor onset followed by a rapid termination upon resolution or cessation of stress exposure. Unnecessary activation of stress responses overexposes corticolimbic brain regions to the neuroplastic and neurotransmission effects of biomediators. Children who have experienced significant adversity show divergence in their stress reactivity patterns [3]. Some have higher basal activation and hypoactivity to acute stress exposure in one or multiple signalling systems, while others show hyperactivity and impaired termination. Elevated inflammation is also a common finding that is a purported mechanism of long-term adverse health and functional outcomes in chronic stress populations [32], the degree of which is different to that of an inflammatory response to infection. Acute stress reactivity to psychological stimuli has been used in a variety of pediatric populations to predict stress-related risks for health and wellbeing [64,111,114–118] and it is generally considered that any deviation (hypoactivation or hyperactivation), is likely indicative of dysfunctional signalling and regulation [16,58].

Few studies have examined stress reactivity patterns in childhood illness populations or considered the role of stress regulation in long-term health and function, despite the significant psychological distress in addition to the direct physiological effects of medical treatments. This is even more surprising considering that many childhood illnesses require the use of synthetic glucocorticoids for treatment. Thus, little evidence for the chronic effects of stress exposure exists in childhood cancer, although some studies have reported that elevated inflammation and oxidative stress persists post treatment [31,102]. Kennedy (2005) found total antioxidant capacity to be lower in ALL patients 6 months post-treatment, and this was correlated with better clinical outcomes including lower rate of infection and hospitalisation, higher quality of life and better treatment tolerance [31]. Similarly, Mazur et al. (2004) found elevated circulating cytokines, TNFα, IL-2 and IL-8, in ALL patients 3, 6 and 12-months post-treatment [32]. Importantly, studies of adult patients have shown a relationship between cytokine status and neurocognitive function [35] providing support for a link between physiological activation and cognitive-affective capacities. However, no research has explicitly investigated physiological stress response profiles, which might provide clues into mechanisms of adverse long-term and late effects in this population. As altered acute stress profiles and biochemical signalling is considered the mechanism linking stressful experiences to long-term health and dysfunction in other populations experiencing significant stress, this is an important area for further research in childhood cancer patient and survivor populations that may provide insight into late effects of childhood cancer [119].

#### **6. Target Organ Activation**

The culmination of stress responses is in allostatic processes preparing the body for a threat and defending homeostasis in the face of changing environmental conditions. However, what was an adaptive response for physical stressors evolutionarily may not be effective for psychological stressors of contemporary lives. The inappropriate activation of stress systems is thought to contribute to a wide range of illnesses reflecting allostatic load [55].

Allostatic load, the aggregation of these subclinical issues or progression of a subclinical issue into a clinical issue over time, is associated with significant risk of morbidity and mortality [3,120]. Children who experience significant life stress have higher risk of heart disease, diabetes, cancer, chronic lung disease, skeletal fractures, liver disease, mental distress disability and overall worse health ratings [17,121]. At the tissue level, evidence of oxidative stress including cellular aging [122–124] and shortened telomeres [125,126] indicate systemic tissue disruption by early life stress.

The consequences of early life stress and chronic stress is further demonstrated by the higher rates of morbidity, as cellular vulnerabilities result in dysfunction, ultimately increasing risk of all-cause mortality in these populations [5]. These same issues are common side-effects observed in children's cancer treatment, especially those treated with synthetic glucocorticoids [28,60,103].

In line with other populations of children who have experienced significant stress, childhood cancer survivors have also been reported to have shortened telomeres, and this has been associated with higher chronic inflammation [104] and higher incidence of late effects during survivorship [105]. Pediatric cancer survivors differ from age-related controls in terms of activation of the adaptive immune system, chronic, low-grade inflammation, as well as immune tolerance resulting from the synthesis of immunomodulators via the tryptophan-kynurenine metabolic pathway [127]. These changes resemble an aging phenotype observed in older populations [128] and are indicative of allostatic load [127]. Some research shows that pediatric cancer survivors have increased biological age relative to their chronological age, as indicated by shortened telomeres [104,105], epigenetic age acceleration [106] and biochemical and molecular markers such as inefficient oxidative phosphorylation, increased lipid peroxidation and decreased expression of metabolic proteins and those involved in mitochondrial biogenesis [107]. Childhood cancer survivors also report an increased incidence of premature frailty associated with radiation treatment [108]. The cytotoxic nature of childhood cancer treatments can cause significant damage and disruption to developing organ systems, which may present more inherent vulnerability to stress exacerbation than the general population. Childhood cancer patients have a higher risk of many chronic illnesses including cardiovascular disease, secondary cancer, metabolic conditions, depression and anxiety, as well as subclinical but lower reported health related quality of life [24,28,60,101]. Dysrhythmias and other indicators of cardiac dysfunction and conductive symptoms are reported in adult survivors of childhood cancers [59], which is most commonly attributed to anthracycline exposure used for anti-neoplastic treatment [129], or radiation to a field that involves the heart. In a population of children and youth who survived mixed types of cancer, 28.2% were reported to exhibit hypertensive or pre-hypertensive signs [130]. Similarly, Cardous-Ubbink (2010) found increased risk of hypertension in adult survivors of childhood cancer, related to BMI, cyclophosphamide, cisplatin or abdominal radiation [33]. In a population of adult survivors of childhood cancer, stress and distress were associated with adverse cardiovascular health conditions such as hypertension, dysrhythmia, dyslipidemia and metabolic syndrome [131]. Even though stress reactivity and function of the systems involved in stress responses have not been investigated in childhood cancer patients or survivors, many of the conditions experienced by this population over their lifetime are those that can be developed or worsened by dysfunctional stress regulation [3,28,60].

It is not possible to differentiate between the contribution of stress dysregulation of target organs over and above that caused by direct cytotoxicity of anti-neoplastic treatment. However, it is still relevant to reducing the burden of illness, as stress dysregulation effects on tissue integrity and function are self-propagating and many adverse health and functional outcomes worsen with time since treatment in childhood cancer survivors. Thus, it is possible that these work in concert to contribute to adverse late-effects, with direct effects of treatment producing vulnerabilities and initial weakness, and stress and other long-term pathways contributing to worsening of function with time. For example, direct disruption to corticolimbic brain regions mediating upstream stress regulation can be further exacerbated by altered stress signalling and may be important targets for intervention to promote better health and function during survivorship.

#### **7. Considerations for Interventions**

Each step in the stress regulatory pathway can alter the function of the system acutely and over time influence the potential of stress to contribute to adverse health and dysfunction. Potential intervention options to reduce the burden of childhood illness centre around

stress-buffering behaviours and may include encouraging social connection [132,133], prosocial behaviour [133] and physical activity [75,134,135], while teaching effective coping strategies. Mutable individual factors related to disposition and behaviour can also have a significant impact on acute physiological activation to a stress stimulus and the effects of stress on long-term health and function. The mutable factors, such as intrapsychic coping strategies, pro-social behaviour and physical activity habits, should be considered clinically meaningful as it relates to any contribution of stress dysregulation on long-term health and function [136]. Programs promoting social connections and development can be expected to have psychosocial and psychobiological benefits through positive changes to cognitive-affective processing and neurological triggering. Expectations of negative social judgement reliably provoke physiological stress responses [65,66]. Strong social connections may reduce negative expectations during cognitive-affective appraisal, reducing physiological activation of stress systems [132]. Further, social support has a strong influence over acute stress reactivity due to the release of oxytocin in the brain, inhibiting CRH production, thereby reducing neurological triggering of the HPA axis and subsequent physiological and affective experiences of a given stressor. Outcomes of social programs in clinical populations rarely focus on clinical indicators, however, positive effects of social support are reported improve stress management [132,133,137].

Several studies have shown that parents of children with cancer have a higher incidence of post-traumatic stress disorder and related symptoms when compared to parents of healthy children [138–140], and that these symptoms were associated between the parents and their children [139]. This suggests that the parents' stress of having a child with cancer may have consequences for the children themselves. Parental stress has been found to be a significant predictor of functional impairment in childhood cancer survivors [141], and childhood cancer survivors may experience different parenting styles including parental overprotection due to stress [142,143]. However, these findings are not conclusive as some research has suggested parenting styles are not different from children without a history of serious illness [144]. These findings indicate the need for further investigation and potentially psychological interventions in childhood cancer patients and parents alike.

Programs promoting physical activity can be expected to have neurobiological and psychobiological benefits through positive changes to cognitive-affective processing, neurological triggering, as well as stress signalling and impacts on target organs [134,135]. Physical activity and fitness both have adaptive effects on stress reactivity and can influence the stress regulatory pathways at multiple steps. Acutely, activity promotes positive mood, reduces negative affect and alters dopamine-GR signalling [75]. Chronically, physical activity promotes executive function, increases neurogenesis of the hippocampus and reduces inflammation as well as promotes healthy function of many of the organ systems of allostatic responses. Together, the effects of physical activity can be expected to reduce acute activation, promote habituation to future stressors and counteract adverse effects of allostatic load on target organ systems [145,146].

Treatment for childhood cancer must prioritise the eradication of the cancer itself; however, secondary considerations must be given to reducing burden of illness during survivorship and improving quality of life and function. To this end, interventions that support stress buffering behaviours may have a beneficial impact on childhood cancer patient and survivors. While clinicians may already promote these types of support for their patients, understanding that these benefits not only improve experiences and subjective quality of life but are likely to have clinical implications for long-term health and wellbeing is critical to ensuring that they are included in holistic treatment of children's cancer.

#### **8. Clinical Implications and Future Research**

Stress regulation is integral to how we navigate dynamic environments in everyday life. Stress, homeostasis and allostasis are concepts that have been developed for decades, with recent attempts to quantify these states being rooted in the study of thermodynamics [147]. The thermodynamic entropy-based stress model proposes that adverse health

states are caused by positive stress entropic load, while negative stress entropic load leads to a protective health state, leading to the idea that energy balance may be a crucial intervention for chronic disease [147]. The chronic or inappropriate activation of stress regulatory signaling or target organ activation contributes to wear and tear on critical organ systems and can contribute to adverse health and functional outcomes [3,8]. Even though neurological changes caused by biomediator feedback occur during excessive or prolonged stress signaling, disruption to the systems regulation can occur at any step in the pathway due to self-regulatory nature. There is evidence that childhood cancer patients may have disruption to several steps in the stress-regulatory pathway including cognitive-affective function, neurological disruption to stress regulatory brain regions, altered adrenal and endocrine function, and disrupted tissue integrity, as well as lower engagement in positive coping behaviours such as physical activity and pro-social habits. Childhood cancer patients experience an array of adverse late effects of their cancer that may be brought on by or exacerbated by dysfunctional stress regulation and adversely affect their physical and mental health. Stress regulation may be a valuable lens through which to examine these chronic morbidities in childhood cancer populations. Further research is needed to better understand acute stress reactivity and stress signaling, as well as the connections between different pathways (i.e., cognitive-affective function and stress outcomes). Larger cohort studies may be necessary to accommodate interindividual variability in stress impacts on individuals and the nature of dysfunction. Even though it is not possible to differentiate between direct and indirect effects of cancer treatment during childhood, a better understanding of how neurological, physiological and psychological disruptions during the experience of childhood cancer interact to produce late effects is important. As treatments continue to improve survival rates in this pediatric clinical population, an emphasis on understanding how to improve health and wellbeing during survivorship has emerged. Clinical recognition of stress as a model during treatment, understanding clinical implications of programs supporting positive coping behaviours—psychological, social, physical activity, may thus be timely.

**Author Contributions:** Conceptualisation, G.E.W. and G.D.W.; methodology, G.E.W. and G.D.W.; investigation, G.E.W., G.D.W., P.C.N. and H.J.; data Curation, G.E.W., J.E.C., K.R., V.M. and G.D.W.; writing—original draft preparation, G.E.W.; writing—review and editing, G.E.W., J.E.C., K.R., P.C.N., S.G.R., H.J. and G.D.W.; supervision, G.D.W.; project administration, G.D.W.; funding acquisition, G.D.W. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by The Exercise Medicine Fund at the Hospital for Sick Children.

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

#### **References**


**Margarita Zaytseva 1,\*, Ludmila Papusha <sup>1</sup> , Galina Novichkova <sup>1</sup>and Alexander Druy 1,2**


**Simple Summary:** The current trend in neuropathology directs to the integrated histo-molecular approach. The traditional concept of histological grade should be complemented by comprehensive diagnostics with the mandatory use of molecular genetic markers. As a consequence, basic types of CNS tumors fall into multiple nosological entities that can be morphologically similar while having fundamentally different pathogenesis and clinical presentation. This trend is particularly evident for ependymal tumors, which harbor molecular markers of decisive importance for the prognosis. This minireview emphasizes recent achievements in ependymoma biology research closely connected with state-of-the-art diagnostics.

**Abstract:** Ependymomas are among the most enigmatic tumors of the central nervous system, posing enormous challenges for pathologists and clinicians. Despite the efforts made, the treatment options are still limited to surgical resection and radiation therapy, while none of conventional chemotherapies is beneficial. While being histologically similar, ependymomas show considerable clinical and molecular diversity. Their histopathological evaluation alone is not sufficient for reliable diagnostics, prognosis, and choice of treatment strategy. The importance of integrated diagnosis for ependymomas is underscored in the recommendations of Consortium to Inform Molecular and Practical Approaches to CNS Tumor Taxonomy. These updated recommendations were adopted and implemented by WHO experts. This minireview highlights recent advances in comprehensive molecular-genetic characterization of ependymomas. Strong emphasis is made on the use of molecular approaches for verification and specification of histological diagnoses, as well as identification of prognostic markers for ependymomas in children.

**Keywords:** ependymoma; risk stratification; molecular group; prognosis

### **1. Introduction**

Ependymal tumors (ependymomas, EPNs), a common type of malignant neoplasms of the central nervous system (CNS), constitute about 10% of all intracranial tumors and about 20% of spinal cord tumors. EPNs rank third in the prevalence of pediatric CNS tumors (after glial and embryonal tumors) [1]. Despite the use of advanced protocols that include maximal safe surgical resection followed by localized radiotherapy, the mortality remains high due to frequent relapses explained by the strong metastatic potential of EPNs complemented by an efficient spread of metastases with cerebrospinal fluid.

Adverse predictors for EPNs are early age at onset, residual tumor tissue after resection, and metastatic lesions in CNS [2–5]; however, the detailed prognosis for EPNs is often hampered by (1) clinical and morphological diversity of the tumors and (2) complex relations of histopathological grades with the prognosis [6,7]. In line with the modern trends in neuropathology, the aggressiveness of a tumor and, accordingly, the prognosis is mainly determined by molecular-genetic aberrations, whereas the conventional, histologically

**Citation:** Zaytseva, M.; Papusha, L.; Novichkova, G.; Druy, A. Molecular Stratification of Childhood Ependymomas as a Basis for Personalized Diagnostics and Treatment. *Cancers* **2021**, *13*, 4954. https://doi.org/10.3390/ cancers13194954

Academic Editors: Saurabh Agarwal and Jianhua Yang

Received: 7 September 2021 Accepted: 29 September 2021 Published: 1 October 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/).

defined grade becomes subsidiary [8,9]. For pediatric EPNs, the relevance of molecular stratification is especially obvious.

Over recent years, the diagnostics of CNS malignancies has been significantly reconsidered. The accent has been shifted from pathomorphology to molecular profiling and the search for clinically informative markers that would justify the selection of a particular therapy. Molecular framework-based stratification schemes have been developed and introduced into clinical practice for a number of CNS tumors; examples include *IDH1/2* mutations and 1p/19q codeletions for gliomas and oligodendrogliomas [10]; *KIAA1549- BRAF* fusions, *MYB*/*MYBL* rearrangements, recurrent pathogenic mutations in *BRAF* and *H3F3A* for pediatric astrocytomas [11,12]; and four molecular groups with the account of *MYC*/*MYCN* amplification for medulloblastomas [13].

EPNs of different molecular etiologies occupy distinct anatomical compartments within CNS. Recurrent genetic or epigenetic alterations found in EPNs are invariably linked to tumor localization. Molecular subgrouping of EPNs is superior to histopathological grading based on the WHO criteria [14]. Gene expression signatures and related subgrouping have shown the highest prognostic value among other studied molecular criteria. A tumor retains its affiliation to a particular subgroup indefinitely (it cannot be switched during progression and/or relapse of the disease), which increases its clinical significance [5,14,15]. An advanced EPN classification has been recently proposed by the Consortium to Inform Molecular and Practical Approaches to CNS Tumor Taxonomy (cIMPACT-NOW) update 7, aimed at connecting localization-dependent molecular groups with tumor progression modes and outcomes [16]. This view has been supported by WHO experts and reflected in the summary of the upcoming fifth edition of the WHO Classification of Tumors of the Central Nervous System (WHO CNS5) [9]. According to the newest CNS tumor nomenclature, ependymomas are subdivided into supratentorial (ST-EPNs), infratentorial (a.k.a. posterior fossa ependymomas, PF-EPNs), and spinal (Sp-EPNs) by localization of the primary tumor; these groups are further stratified by (epi)genetic features.

#### **2. Molecular Profiles of ST-EPNs**

ST-EPNs are fairly rare and show considerable genetic heterogeneity. ST-EPNs have been recently stratified into two major groups: supratentorial ependymoma, *ZFTA* fusionpositive (ST-EPN-ZFTA) and supratentorial ependymoma, *YAP1* fusion-positive (ST-EPN-YAP1) [9] consistently with gene expression and/or DNA methylation signatures revealed by transcriptomic methods and/or whole-genome DNA methylation profiling, respectively.

#### *2.1. ST-EPN-ZFTA Group*

Gain-of-function rearrangements in *ZFTA* or *YAP1* are specific for ST-EPNs. At that, ST-EPN-ZFTA tumors are prevalent (50–75% and 25% of ST-EPNs in children and adults, respectively [7,14,17–20]), while ST-EPN-YAP1 tumors are rare (3–10% in different cohorts [7,14,18,19,21,22]). The archetypal chimeric transcript harbored by *ZFTA*-rearranged ependymomas is *ZFTA*–*RELA*, hence the ST-EPN-RELA is a traditional designation for this group [14]. Alternative *ZFTA* fusions (non-*RELA*, e.g., *ZFTA-NCOA1*, *ZFTA-NCOA2*, *ZFTA-MAML2* [23–28], and *MN1-ZFTA* [28]) are less common.

Recurrent *ZFTA*–*RELA* fusion is a unique molecular hallmark of *ZFTA*-positive EPNs not found in other CNS tumors. Nine different transcript variants have been described, differing by breakpoints in *RELA* and its partner gene; the prevalent isoform comprises *ZFTA* exon 2 spliced to *RELA* exon 2 [17,22,29]. Formation of the *ZFTA*–*RELA* intrachromosomal gene fusion results from multiple double-strand breaks in 11q13.1 with subsequent random reassociation (typical for chromotrypsis); hence the diversity of fusion points for such transcripts. The oncogenic impact of classical *ZFTA–RELA* fusions was elucidated in recent studies [30,31].

*RELA* encodes the RelA (p65) subunit of the dimeric nuclear factor-κB (NF-κB), most known as a master regulator of immune responses and inflammation. NF-κB promotes apoptosis inhibition, cell growth, and pro-angiogenic signaling—the basic components of oncogenesis and tumor progression. Expression of *RELA* chimeras results in constitutive activation of NF-κB signaling pathway [17] and associated resistance of the tumor to chemo and radiation therapies [32]. ZFTA chimeric proteins accumulate in the nucleus. A zinc finger domain in the truncated ZFTA protein endows the chimeras with extraordinary high affinity to DNA. The oncoprotein interferes with chromatin structure at ST-EPN-associated loci, enabling the RELA transactivation domain to induce their transcription [30]. Moreover, apart from the canonical NF-κB pathway activation, *ZFTA*–*RELA* fusions may trigger other gene expression programs through recruitment of transcriptional co-activators BRD4, EP300, and CBP, which participate in chromatin-related pathways and represent potential druggable targets [31,33].

EPNs with the *ZFTA* gene fused with a non-*RELA* partner gene are considerably less common. These tumors have variable histological structures and, apart from the ependymomal component, may additionally involve pleomorphic xanthoastrocytoma-like, astroblastoma-like, malignant teratoma-like, embryonal tumor-like, or sarcoma-like patterns. Despite the heterogeneous morphology, these tumors are (epi)genetically similar and tend to resemble the classic *ZFTA*–*RELA*-fused EPNs, as revealed by methylome assay. A detailed analysis of DNA methylation profiles allows subdivision of these tumors into two clusters, one of them comprising tumors with histological features of astroblastomas and xanthoastrocytomas, harboring *ZFTA*–*MAML2* and *MN1*–*ZFTA* rearrangements; the second cluster comprises tumors histologically resembling small-cell sarcomatoid carcinomas and undifferentiated sarcomas, harboring *ZFTA*–*NCOA1* and *ZFTA*–*NCOA2* rearrangements [24–28].

The presence of recurrent *ZFTA*–*RELA* fusions has been repeatedly implicated as an adverse prognostic factor [5,14,18]. Five-year rates of event-free survival (EFS) and overall survival (OS) for ST-EPN-ZFTA tumors never exceed 29% and 75%, respectively [14]. Within the ST-EPN-ZFTA group, additional risks of relapse have been associated with 1q gains [5]. Interestingly, the St Jude Young Children 07 (SJYC07) study (encompassing ependymal tumors diagnosed in <3-year-olds) identified similar 4-year EFS rates for ST-EPN-ZFTA, ST-EPN-YAP1, and PF-EPN group A [7]. Consistently, two other studies conducted independently by the Italian Association of Pediatric Hematology and Oncology AIEOP and the Children's Oncology Group (trial ACNS0121) revealed no difference in survival rates for ST-EPNs with and without *ZFTA*–*RELA* fusion [4,34].

*ZFTA* (non-*RELA*)-fused EPNs have an especially dismal prognosis, with EFS rates significantly lower compared with classical *ZFTA*–*RELA*-fused EPNs, while the corresponding OS rates are comparable [26]. However, these findings are preliminary, given the small number of cases reported so far. In the context of ST-EPN heterogeneity, it might be useful to consider *ZFTA* fusions with atypical (non-*RELA*) partners as a distinguishing feature for a separate group, the prognostic and clinical relevance of which is yet to be specified.

#### *2.2. ST-EPN-YAP1 Group*

ST-EPN-YAP1 tumors show an aberrant activity of transcription co-activator YAP1 (Yes-associated protein 1) related to its abnormal accumulation in the nucleus. With YAP1 being a direct regulator of TEAD and SMAD transcription factors, its escape from Hippo-dependent sequestration through accumulation in the nucleus results in sustained proliferative signaling via WNT and Hedgehog. More accurately, the nucleus accumulates the oncogenic fusion protein YAP1–MAMLD1 transferred from the cytoplasm to the nucleus independently of its YAP1-Ser127 phosphorylation status that limits the nuclear import of YAP1 in normal cells [35]. Apart from the prevalent *YAP1*–*MAMLD1* fusions, ST-EPN-YAP1 may harbor relatively rare structural variants, e.g., *YAP1*–*FAM118B* [14,36]. In some cases, the formation of *YAP1* fusions involves focal copy number alterations mapping to the 30 portion of the gene (11q22.1–11q21.2) [37]. Unlike *ZFTA*-positive ependymomas, ST-EPN-YAP1 tumors have balanced genomes with local aberrations in the *YAP1* locus and no evidence of chromothripsis.

Andreiuolo et al. (2019) reported a multicenter retrospective study on what is so far the largest cohort of patients with *YAP1*-positive EPNs (*n* = 14). Overall survival for these patients (median observation time of 4.8 years within the range of 0.6–16 years) constituted 100%. It is important to note that the boy-to-girl ratio for the studied cohort was 1:6.5, and only three of the patients were over three years old at the time of diagnosis (the median age at diagnosis constituted 8.2 months) [37]. The best survival rates for the ST-EPN-YAP1 group among other EPNs were also reported by other authors [7,14]. Careful de-escalation of conventional EPN treatment protocols specifically for ST-EPN-YAP1 patients is currently under scrutiny. An opportunity to exclude (delay or dismiss) radiation therapy alleviates the risks of severe cognitive dysfunctions, endocrinopathies, and secondary tumors [38].

#### *2.3. Non-ZFTA/Non-YAP1 ST-EPNs*

The molecular diversity of ST-EPNs exceeds the currently established ZFTA-YAP stereotype. Tumors with neither *ZFTA* nor *YAP1* alterations are considered as a separate group, and recent findings emphasize the need for a finer specification. A distinct entity is formed by *PLAGL1* rearranged EPNs, harboring *EWSR1*–*PLAGL1* and less commonly *PLAGL1*–*FOXO1* or *PLAGL1*–*EP300* fusions [39], which echoes molecular landscapes of soft tissue sarcomas and a group of rare mesenchymal (non-meningothelial) and glioneuronal CNS tumors with *EWSR1*–non-ETS fusions [40,41]. Nevertheless, for the vast majority of ST-EPNs lacking recurrent chromosomal rearrangements, the oncogenic driver events remain elusive. Several reports reveal the presence of fusion genes *MAML2*–*ASCL2*, *MARK2*– *ADCY3* [19], *PTEN*–*TAS2R1* [14], *PATZ1*–*MN1*, *MYH9*–*SEC14L2*, *MTMR3*–*NCOA3* [24], *TMEFF2*–*FOXO1*, *PCGF1*–*CREBBP* [20], *FOXO1*–*STK24*, as well as *EP300*–*BCORL1* in such tumors [21]. Olsen et al. (2015) described two cases of hemispheric infantile EPN-like gliomas with *ALK* fusions (*CCDC88A*–*ALK* and *KTN1*–*ALK*), both of them morphologically ambiguous: the tumors showed glial phenotypes and resembled glioblastomas [42]. In the summary of the upcoming WHO CNS5, such tumors have been reclassified and renamed as infant-type hemispheric gliomas harboring receptor tyrosine kinase gene rearrangements [9]. Torre et al. (2020) reported in-frame fusions *AGK*–*BRAF* and *MYO5A*–*NTRK3* as potential targets for therapeutic inhibition [20]. In sum, these observations indicate the absence of a single driver mechanism for this group of tumors while underscoring the importance of their distinction from other CNS neoplasms.

#### **3. Molecular Profiles of PF-EPNs**

PF-EPNs, more prevalent in children than adults, constitute about 2/3 of intracranial ependymal tumors of childhood. Based on high-throughput molecular techniques, PF-EPNs are subdivided into two molecular groups: PF-EPN group A (PF-EPN-A), and PF-EPN group B (PF-EPN-B) [9].

#### *3.1. PF-EPN-A Group*

This highly heterogeneous group comprises 85–90% of infratentorial EPNs. PF-EPN-A tumors are often located laterally within the posterior fossa and occur predominantly in infants and young children, twice more frequently in boys than in girls; the average age at diagnosis constitutes 3.5 years [14]. The patients are at high risk of relapse, even under multimodal therapy and in the absence of extra adverse prognostic markers [5]. Identification with PF-EPN-A represents a strong independent prognostic factor associated with the worst rates of survival. According to Zapotocky et al. (2019), 5-year and 10 year EFS for PF-EPN-A constitute, respectively, 43% and 37% [3]. Relapses are typical for PF-EPN-A but not PF-EPN-B and, consistently, 10-year OS rates for PF-EPN-A are significantly lower than for PF-EPN-B (56–58% vs. 88–100%) [4,14,15]. Additional negative clinical predictors for PF-EPN-A are the presence of residual tumor tissue (incomplete resection) and adjuvant radiation therapy refusal [3]. Retrospective evaluation of outcomes for the patients receiving treatment under HIT-2000 protocol implicated residual tumors, 1q gains, and high mitotic activity of tumor cells (>10 mitotic figures per 10 fields of

view) as independent adverse predictors for PF-EPNs in general and PF-EPN-A tumors in particular [2]. Cytogenetic prognostic factors for PF-EPNs include 1q gains and 6q losses [43,44]. These cytogenetic abnormalities are detected in 18.9% and 8.6% of PF-EPNs, respectively. At that, the 1q gained PF-EPNs harbor 6q losses at an increased frequency of 17.7% [43]. Both types of copy number variations have been qualified as adverse predictors. Five-year progression-free survival rates were 50% for EPNs without 1q gain and 6q loss, as opposed to 32% for 1q gain only, 7.3% for 6q loss only, and 0% for both 1q gain/6q loss tumors [43]. The ultra-high risks conferred by the co-occurrence of cytogenetic markers in PF-EPN-A patients should be taken into account for the treatment regimen optimization.

PF-EPN-A tumors reveal characteristic aberrant patterns of DNA methylation, the so-called CpG-island methylator phenotype (CIMP) with extensive hypermethylation of CpG-islands in promoter regions of multiple genes. This effect critically interferes with the function of PRC2 (Polycomb repressive complex 2) [45,46]—a transcription repressor protein complex that facilitates methylation of nucleosome histone H3 at amino acid residues H3K27 and H3K9 thus inhibiting the expression of key regulatory genes responsible for cell fate determination and differentiation. Bayliss et al. (2016) revealed the deficiency or complete loss of H3K27me3 in PF-EPN-A tumors [46]. This finding complements the earlier hypotheses on the central role of epigenetic mechanisms in PF-EPN pathogenesis inferred from the absence of presumably pathogenic mutations in chromatin remodeling genes and enzymes that catalyze post-translational modifications (e.g., methylation) of histones in whole-genome sequencing data [17,45,46]. The recruitment of Polycomb group (PcG) transcription repressors to chromatin requires the presence of non-methylated CpGs; accordingly, the loss of H3K27me3 methylation has been associated with dense hypermethylation of CpG-islands preventing the recruitment of PcG proteins to chromatin by steric hindrance.

An advanced investigation of molecular mechanisms responsible for the observed epigenetic malfunctioning revealed a plausible association of the H3K27me3 deficiency with elevated expression levels of accessory proteins encoded by *EZHIP* (formerly *CXorf67*) and *EPOP* (formerly *C17orf96*) [47–49]. As demonstrated by Hübner et al. (2019), EZHIP is a competitive inhibitor of PRC2. A conservative stretch of amino acids in the C-terminal portion of EZHIP mimics the K27 methylation target in histone H3, albeit with K27M substitution. The binding of methionine M27 (instead of lysine K27) to the active center in the histone-lysine N-methyltransferase subunit of PRC2 blocks its catalytic activity [50]. Somatic missense mutations in *EZHIP* are detected in a small proportion of PF-EPN-A tumors (<10%) [48]. Jain et al. (2019) demonstrated that such mutations have no influence on H3K27me3 levels thus disproving their functional significance [51]. Noteworthy, no loss-of-function mutations in *EZHIP* (nonsense substitutions or frameshift indels) have been reported. Elevated expression of *EZHIP* in tumors may be caused by mutations in *cis*-regulatory elements; the same effect may be conferred by the formation of fusion genes involving *EZHIP* locus (for instance, *MBTD1*–*EZHIP* fusion described for low-grade endometrial stromal sarcoma [52]). However, no fusions comprising *EZHIP* or PRC2 subunit-encoding genes (e.g., *EED*, *SUZ12*) have been described for EPNs.

Related signatures of disrupted epigenetic regulation have been associated with *H3* K27M mutations typical for diffuse midline gliomas (DMGs) but rarely found in PF-EPN-A (<5% of the cases). Noteworthy, in EPNs such mutations are harbored by canonical histoneencoding genes *HIST1H3C* and *HIST1H3B*, whereas in DMGs they are predominantly found in a replacement histone gene *H3F3A* (90% of the cases) [48,53–55]. Given the mutually agonistic roles of the onco-histone H3 K27M and EZHIP, it would be natural to expect similar patterns of disease progression and therapy outcomes for *H3* K27M-mutant DMG and EZHIPhigh PF-EPN-A. Indeed, in DMG, disruption of H3K27me3-mediated epigenetic regulation is associated with an extremely aggressive course of the disease, typically presenting with sustained tumor growth and polychemotherapy resistance [56–58]. Similarly, effective chemotherapy regimens for PF-EPN-A are missing [59] and therapeutic options for relapses are extremely limited [5,60–63].

Despite the uniformity of methylation profiles within PF-EPN-A, tumors of this group show considerable molecular heterogeneity and can be additionally classified into two major subgroups A1 and A2 (and ultimately into nine minor subtypes by using additional markers: gains 1q, deletions 22q, 6q, and 10q, and OTX2 protein expression). PF-EPN-A1 tumors are distinguished by pronounced expression of the homeotic HOX genes (*HOXA1/2/3/4*, *HOXB2/3/4*, *HOXC4*, and *HOXD4*) which define the segmental (rhombomeric) organization of the hindbrain in early embryogenesis. PF-EPN-A2 tumors hyperexpress *EN2*, *CNPY1*, and *IRX3*—a group of genes involved in the rhombomere differentiation. Expression of A1- and A2-specific genes within the developing hindbrain shows distinct zonality—increased expression of A2 markers is characteristic of the rostral portion at the border with the midbrain, while expression of HOX genes is more pronounced in caudal segments of the brainstem and spinal cord. Differential expression of the spatial patterning genes in A1 and A2 tumors apparently reflects their origin from different hindbrain structures. However, the practical relevance of the advanced A1/A2 subgrouping is questionable. Stratification by clinical factors (gender, age at diagnosis, tumor resection volume, and received therapy) revealed no significant differences between A1 and A2 tumors, except the patterns of relapse (PF-EPN-A1 tumors more often produce local than distant relapses, and vice versa) [48].

#### *3.2. PF-EPN-B Group*

In contrast to PF-EPN-A tumors which predominantly affect children, PF-EPN-B tumors are more common in adults. In adolescents (aged 10–17), about 45% of newly diagnosed EPNs fall into this group. The prognosis for PF-EPN-B tumors is favorable: 10-year OS rates for the patients after subtotal and gross total resections reach 66.7% and 96.1%, respectively [3,14,15,64]. Thus, the prognosis for this group strongly depends on the extent of surgical resection. The occurrence of delayed relapses (10 years after the onset) underscores the importance of long-term follow-up [64]. Patients with R0 may benefit from chemo- and radiation-sparing strategies; such possibility is being considered [38]. The observed difference in patterns of recurrence between PF-EPN-A and PF-EPN-B adds to the relevance of comprehensive molecular characterization of a tumor as early as possible.

By now, recurrent mutations or fusion genes in PF-EPN-B tumors are missing, and no clear drivers for this group have been identified. Ciliogenesis and microtubule assembly are deregulated only in PF-EPN-B tumors, while several canonical cancer-associated pathways operate in the PF-EPN-A group (VEGF, PDGF, EGFR, RAS signaling, etc.) [14]. PF-EPN-B tumors harbor major cytogenetic aberrations including gains 1q, monosomies 6, 10, and 17, trisomies 5, 8, and 18, and deletions 22q [64]. The diversity of cytogenetic profiles revealed for PF-EPN-B indicate inherent genomic instability and suggest that these tumors emerge from multiple driving events. Similarly with PF-EPN-A, the PF-EPN-B group shows significant heterogeneity, with distinct molecular subtypes of different demographics, copy number alterations, and gene expression signatures. By contrast with PF-EPN-A, gains 1q pose no extra risks for PF-EPN-B tumors. Losses 13q may represent a more reliable negative prognostic marker than gains 1q; however, this assumption requires further substantiation, particularly as the basis for de-escalation of therapy regimens. However, the extent of resection remains the strongest predictor of poor outcomes for this group. Given the patient data scarcity, advanced stratification within PF-EPN-B remains clinically irrelevant [64].

#### *3.3. ST-EPN-ZFTA-like PF-EPNs*

Unique cases of *ZFTA*–*MAML2*, *ZFTA*–*RELA*, and *ZFTA*–*NCOA2* fusion in PF-EPN were reported recently. These tumors revealed characteristic *ZFTA*-mediated gene expression and whole-genome DNA methylation signatures corresponding to the ST-EPN-ZFTA group; accordingly, they were classified as "ST-EPN-ZFTA" despite the infratentorial localization [65].

A summary of the intracranial EPN classification is given in Figure 1.

**Figure 1.** Basic classification of intracranial ependymal tumors.

#### **4. Molecular Profiles of Sp-EPNs**

Sp-EPNs constitute a heterogeneous group with a generally favorable prognosis. These tumors mostly occur in adult patients and are rare in children. Clinical outcomes for Sp-EPNs are better than for intracranial EPNs, with 5-year OS rates within the range of 60–90% [14]. Three molecular groups of Sp-EPNs were originally identified, including subependymomas, myxopapillary Sp-EPNs (SP-MPE), and Sp-EPNs per se; the molecular subgrouping shows excellent concordance with corresponding histopathological subtypes [14]. Our knowledge on the molecular pathogenesis of Sp-EPN tumors is limited. The groups reveal characteristic somatic copy number aberrations; most Sp-EPNs harbor 22q deletions involving neurofibromin 2 (*NF2*) tumor suppressor gene, whereas SP-MPEs show chromosomal instability.

SP-MPEs, the most prevalent type of pediatric spinal cord EPNs, predominantly arise in the conus medullaris, cauda equina, and filum terminale regions [66]. Despite their low mitotic index and slow-growing nature, SP-MPEs generally have far more aggressive behavior than other low-grade CNS tumors. Furthermore, pediatric SP-MPEs are especially aggressive, with much higher rates of local recurrence and secondary seeding to distant craniospinal sites or local spinal sites (64% cf. 32% in adults) [67]. As demonstrated by Ahmad et al. (2021), pediatric SP-MPEs exhibit aberrant activity of the mitochondrial metabolic pathways [68]. The only recurrent focal amplification identified for this group involves *HOXB* gene cluster mapping to 17q. *HOXB13* amplification represents a candidate diagnostic marker for SP-MPEs. The elevated expression of *HOXB13* enhances tumor cell proliferation and dissemination, playing a critical role in the development of metastasis [68]. Due to the high propensity for local recurrence and distant neural axis dissemination, the summary of the upcoming WHO CNS5 identifies SP-MPEs with grade 2 (rather than grade 1). SP-MPEs have distinctive histopathological features such as well-organized papillary architecture, with vascular cores and abundant mucinous extracellular matrix. Histological examination of tumor tissue is necessary and sufficient for the diagnosis of SP-MPE, whereas genetic testing is accessory [9].

A rare subtype of Sp-EPN in adult patients has been described recently, presenting highly aggressive clinical behavior with early metastasis, diffuse leptomeningeal spread throughout CNS, and resistance to standard treatment protocols. All of them harbored *MYCN* amplification and no other recurrent pathogenic events [69–71]. Importantly, these tumors formed a distinct methylation cluster of their own, and none of them clustered with any of the previously identified nine EPN groups. Recognizing the importance of clinical and molecular data on such tumors, the summary of the upcoming WHO CNS5 reports a novel nosological entity of *MYCN*-driven Sp-EPNs with dismal outcomes [9].

Genetic alterations found in particular in EPN groups are summarized in Table 1. Subgroup-specific diagnostic and candidate genes in pediatric EPNs are contained in Table 2.


**Table 1.** Summary of major molecular markers of ependymal tumors.


*Cancers* **2021**, *13*, 4954

**Table 2.**

Subgroup-specific

 diagnostic

 marker and candidate

 genes involved

 in recurrent

chromosomal

abnormalities

 in pediatric

 EPNs.






*Cancers* **2021**, *13*, 4954

**Table 2.** *Cont.*

NA—nonapplicable.

September 2021). \*\*\* Evidence-based variant (nucleotide substitution, copy-number variation, and fusion) of listed genes categorization based on the Joint Consensus Recommendation of the Association for Molecular Pathology, American Society of Clinical Oncology, and College of American Pathologists (AMP/ASCO/CAP recommendations). \*\*\*\* Potential roles of the cancer hallmark genes are annotated using COSMIC Cancer Gene Census (https://cancer.sanger.ac.uk/census, accessed on 20 September 2021), GeneCards: The Human Gene Database (https://www.genecards.org/, accessed on 20 September 2021), and KEGG: Kyoto Encyclopedia of Genes and Genomes (https://www.kegg.jp/kegg/, accessed on 20 September 2021).

#### **5. Molecular Profiles of Subependymomas**

Subependymomas are exceptionally rare slow-growing benign neoplasms, accounting for only 0.07–0.7% of all intracranial tumors [14,72]. These tumors typically arise in adults (aged 22–76 years) without strict predilection to a particular site in CNS. The most typical locations, the fourth and lateral ventricles, are encountered in up to 85% of the cases, followed by septum pellucidum, brainstem, and spine [73]. DNA methylation profiles of subependymomas differ depending on tumor localization, which provides certain grounds for molecular subgrouping [14]. However, all subependymomas have a favorable prognosis independently of localization. According to the cIMPACT-NOW group recommendations and the summary of the upcoming WHO CNS5, morphological examination provides adequate means for the diagnosis and prognostication; the integrative histo-molecular approach for subependymomas is accessory [9,16]. Recent findings suggest that the process of clonal evolution in subependymomas may give rise to more aggressive tumor clones enriched with pure EPN phenotypes, chromosome 6 losses, and *TERT* mutations. These markers, associated with increased risks of recurrence, should be considered as an indication for more intensive therapies, especially under conditions of subtotal tumor resection [44].

#### **6. Laboratory Approaches for EPN Diagnostics**

From a histological perspective, EPNs show moderate cellularity and variable mitotic activity; they consist of monomorphic rounded or oval cells with scant cytoplasm and vesicular nuclei containing granular (salt-and-pepper) chromatin. Key histological features of EPNs are perivascular (pseudo) rosettes and ependymal rosettes. EPNs are positive for glial fibrillary acidic protein (GFAP), S100, vimentin, rarely cytokeratin, and epithelial membrane antigen (EMA, positive along the luminal surface of ependymal rosettes or as characteristic dot-like or ring-like intracytoplasmic patterns) and negative for most neuronal antigens [6].

No unified standards for molecular diagnostics of EPNs have been introduced so far. The molecular group for each particular case is determined by whole-genome DNA methylation profiling as a golden standard. Comprehensive analysis of DNA methylation signatures is the method of choice for differential diagnosis within a broad spectrum of glial and embryonal tumors including CNS neuroblastoma with *FOXR2* activation, CNS Ewing sarcoma family tumor with *CIC* alteration, CNS high-grade neuroepithelial tumor with *MN1* alteration, and CNS high-grade neuroepithelial tumor with *BCOR* alteration [24,74]. The advantage of using this technique is the suitability of formalin-fixed paraffin-embedded tumor tissue for the analysis (methylated sites in genomic DNA are preserved during fixation, dehydration, etc.). However, the use of DNA methylation assays in routine laboratory practice has serious limitations, as the data processing is complex and the method itself is expensive, sophisticated, and labor-consuming. Alternative diagnostic algorithms may be based on a combination of economically justified methods including histological examination, immunohistochemistry (IHC) testing, reverse transcription-polymerase chain reaction (RT-PCR), Sanger sequencing, fluorescence in situ hybridization (FISH), and probably also the NanoString nCounter® platform (Figure 2).

**Figure 2.** The integrated approach for molecular group determination applied to ependymal tumors.

#### *6.1. Differential Diagnosis of ST-EPNs*

In many cases, unambiguous determination of the molecular group of a tumor can be afforded by a certain combination of routine morphological and genetic approaches [75]. For instance, nuclear accumulation of p65 (RelA) and the presence of *ZFTA*–*RELA* fusion are sufficient for the identification of the sample with the core ST-EPN-ZFTA group harboring the classical *ZFTA*–*RELA* transcript. The diversity of fusion transcripts, which results from the variability of breakpoints in *RELA* and its partner genes, significantly complicates the identification of particular rearrangements and their use as markers. Conventional RT-PCR tests are targeted at the two most frequent fusions *ZFTA* (exon 2)–*RELA* (exon 2) and *ZFTA* (exon 3)–*RELA* (exon 2) [19,75,76].

A highly efficient way of screening for *ZFTA* and *RELA* rearrangements is provided by FISH with break-apart probes for one of the fusion partner genes. This approach allows detecting rearrangement of the gene of interest without accurate determination of the breakpoint [19,21]. Its excellent concordance with other methods including nuclear expression of RelA and DNA methylation profiling should be noted [19].

*ZFTA* gene rearrangements induce the hyperexpression of L1 cell adhesion molecule (L1CAM) and/or cyclin D1 may be considered as a surrogate marker of the ST-EPN-ZFTA group [18,20,22,23]. However, IHC is not sufficient on its own: the results require confirmation by independent alternative methods since neither of the antibodies (anti-p65, anti-L1CAM, anti-cyclin D1) has enough sensitivity and/or specificity to reliably verify the ST-EPN-ZFTA group [20,22]. The diagnosis of ST-EPN-ZFTA with alternative (non-*RELA*) fusion gene can be suspected if tumor cells show immunopositivity for L1CAM and negativity for p65 [26].

Identification of a tumor with the ST-EPN-YAP1 group is based on detection of either *YAP1* rearrangement by FISH or one of the *YAP1*-fusions (*YAP1*–*MAMLD1*, *YAP1*– *FAM118B*) by RT-PCR, while IHC with anti-YAP1 and/or anti-Claudin-1 is noninformative [19,37].

#### *6.2. Differential Diagnosis of PF-EPNs*

Panwalkar et al. (2017) proposed IHC tests for H3K27me3 as a straightforward and affordable method for PF-EPN stratification. The authors demonstrate that PF-EPN-B tumors are completely positive for H3K27me3, whereas the presence of H3K27me3 signal in less than 80% of tumor cell nuclei (against the total positivity of endothelial cells used as an internal control) is indicative of PF-EPN-A. Moreover, groupings based on whole-genome DNA methylation profiling vs. IHC tests for H3K27me3 were 99.1% concordant; the outlier was H3K27me3-negative tumor from a 12-year-old female patient,

classified as PF-EPN-B on the basis of DNA methylation analysis [77]. The methodology was further enhanced by Fukuoka et al. (2018) who attempted at finer quantitation of the IHC data. In about 62% of prediagnosed PF-EPN-A tumors examined by the authors, the H3K27me3 reactivity was shown by 5–50% of the tumor cell nuclei; for the rest of PF-EPN-A tumors (about 40%), the proportion was less than 5%. In contrast, the vast majority of PF-EPN-B tumors showed intact expression of H3K27me3, with 90–95% of tumor cell nuclei stained positive, except two cases of reduced positivity (10–60%) classified as PF-EPN-B by DNA methylation profiling. Overall, the cut-off level of 80% H3K27me3-positive tumor cells allowed distinguishing PF-EPN-B from PF-EPN-A with 100% specificity and 86.7% sensitivity [21].

Antin et al. (2020) suggested complementing the routine IHC panels for CNS tumor diagnostics with anti-EZHIP staining. The intensive diffuse nuclear staining with >90% tumor cells immunopositive was obtained for PF-EPN-A (with the exception of rare *H3* K27M-mutant tumors), diffuse midline gliomas with wild-type (non-mutant) *H3F3A*, and germinomas [78]. Nambirajan et al. (2021) evaluated an extended panel of IHC markers (H3K27me3, acetyl-H3K27, H3K27M, ATRX, EZHIP, EPOP, and Tenascin-C) for the use in differential diagnostics of PF-EPNs. The authors demonstrated that a combination of EZHIP- and H3K27me3-specific antibodies is sufficient for reliable verification of PF-EPN-A; this finding illustrates the consistency between genetic and IHC profiles of a tumor [79].

Given the existence of rare EPN subtypes e.g., PF-EPN with *ZFTA* rearrangement [65] or ST-EPN harboring *ZFTA* fusions with atypical (non-*RELA*) partners, correct identification of a tumor with a particular group requires an extended diagnostic algorithm accounting for gene expression signatures. The creation of a universal diagnostic tool for the determination of molecular subtypes of EPN can be based on the analysis of gene expression signatures using the NanoString nCounter® platform. The assay must be carried out with customized panels of markers, corresponding to EPN group-specific signatures of upregulated genes, designed specifically for this purpose. Lastowska et al. (2021) identified group-specific marker genes for *RELA*-fused EPN (*RELA*, *ELL3*, *FBP2*, *PCP4L1*, and *MYO3A*), *YAP1*-fused EPN (*MRAP*, *IGF1*, *CAPS*, and *WWC1*), PF-EPN-A (*LAMA2*, *ALDH1L1*, *SLC6A13*, *IGSF1*, and *CXorf67*), and PF-EPN-B (*NELL2*, *DNAH1*, *CEP83*, *C9orf72*, and *NXNL2*) tumors. The NanoString-based approach clearly separated PF-EPNs into two clusters, corresponding to the defined PF-EPN groups, based on the expression of selected genes. Extended cluster analysis allowed subdivision of PF-EPN-A tumors into PF-EPN-A1 and PF-EPN-A2 subgroups using four marker genes (*SKAP2*, *WIF1*, and *EN2*, *CNPY1*, respectively). The described gene panel for *RELA*-positive EPN did not allow differentiation of EPN with *ZFTA*–*MAML2* fusion [80]. The potential of the NanoString method for diagnosis and advanced classification of other CNS tumors has been confirmed in several studies [81–83]. The approach provides reliable stratification of medulloblastomas into four groups (designated WNT, SHH, group 3, and group 4) based on the evaluation of 22 transcripts expressed differentially among the groups and contributing to characteristic group-specific gene expression signatures [81,82]. Primitive neuroectodermal tumors of the CNS (CNS-PNETs) have been already defined as four new molecularly defined entities (CNS neuroblastoma with *FOXR2* activation, CNS Ewing sarcoma family tumor with *CIC* alteration, CNS high-grade neuroepithelial tumor with *MN1* alteration, and CNS high-grade neuroepithelial tumor with *BCOR* alteration) using a single multi-gene tumor-specific signature [83]. However, differential diagnostics of EPNs in mixed series with other CNS tumors by the NanoString approach is impossible. For correct molecular stratification of ependymal tumors by the NanoString approach, all samples intended for comparison must be morphologically identified as EPN prior to the analysis.

#### **7. Therapeutic Targeting of EPNs**

Despite the profound insights into ependymal tumor biology, consensus recommendations for the management of patients with EPNs with regard to molecular diagnosis are missing. As tumors are resistant to conventional chemotherapy, the search for drug-

gable targets is highly relevant. However, no candidate targets have been identified despite the extensive efforts in molecular profiling. Clinical studies on general cohorts showed no significant objective response to ERBB1/2 (lapatinib) and VEGF inhibitors (bevacizumab, sunitinib), despite the evidence on overexpression of ERBB2, ERBB4, or VEGF in EPNs [84–87].

In the molecular era, cancer-specific somatic aberrations should be taken into account when choosing a treatment strategy. For PF-EPNs, the pathogenesis of which involves epigenetic mechanisms, the possibility of using pharmaceuticals targeted at epigenetic modifications, including abnormalities of DNA methylation and histone modifications, seems most rational. Despite the promising results of pre-clinical research [88,89], histone deacetylase (HDAC) inhibitors showed no therapeutic activity in patients, apparently due to the intricacies of subcellular localization of HDACs and inability of the inhibitors to accumulate inside the brain tissue in concentrations sufficient for therapeutic response [90]. Nevertheless, the search for new HDAC inhibitors with appropriate brain-penetrating capacities and safety profiles may provide a useful treatment strategy [91]. The possibility of using BET-bromodomain inhibitors as anticancer therapeutics is being investigated in pediatric EPN stem cell models [92]. The upregulation of EZHIP could have important implications for therapeutic approaches. As reported by Han et al. (2020), EZHIP has a conservative PALB2-binding domain which enables its functioning as a competitive inhibitor of BRCA2. Elevated levels of EZHIP prevent the formation of BRCA1-PALB2- BRCA2 complexes thus inhibiting the homologous recombination-mediated DNA repair pathway. The results indicate the potential of PARP inhibitors as targeted therapeutics in PF-EPN-A, especially when combined with radiotherapy [61].

NF-kappa B inhibitors are considered potential therapeutic agents for the treatment of ST-EPN-ZFTA with constitutive activation of the NF-κB signaling pathway. A trial currently in phase II is evaluating the effectiveness of marizomib—a second-generation irreversible proteasome inhibitor, enrolling patients with histologically proven spinal or intracranial EPN, including but not limited to ST-EPN-ZFTA (ClinicalTrials.gov Identifier: NCT03727841, accessed on 20.09.2021). At the moment, the enrollment is complete, and the study is in progress. Specific expression of PD-L1 on both tumor and myeloid cells in ST-EPN-RELA has been demonstrated, accompanied by high levels of PD-1 expressed by tumor-infiltrating T cells (both CD4 and CD8) [93,94]. In the context of immunotherapy, ST-EPN-RELA progression may be controlled with PD-1 inhibitors, such as pembroluzimab or nivolumab [95].

Despite the principal shift in the EPN diagnostics and molecular stratification, its immediate impact on the existing treatment regimens is low. The correction would require preclinical and clinical trials for EPNs with due consideration of the molecular subgrouping.

#### **8. Conclusions**

As demonstrated by advanced studies of the last decade, ependymomas constitute a heterogeneous group of tumors and differ by molecular etiology. This minireview underscores the importance of comprehensive molecular profiling for ependymal tumors aimed at identifying specific expression signatures and/or (epi)genetic variants. Molecular identification of an ependymal tumor with a particular molecular group should follow its anatomical and histopathological assessment. Advanced stratification of patients into risk groups provides a framework for personalized management, e.g., allows de-escalation of the therapy in patients with low-risk tumors (supratentorial ependymomas group YAP1 and infratentorial ependymomas group B). Detailed understanding of causative molecular abnormalities for particular tumors is pivotal for the development of novel therapeutic options.

**Author Contributions:** Conceptualization, M.Z.; writing—original draft preparation, M.Z., L.P. and A.D.; writing—review and editing M.Z., L.P., G.N. and A.D.; supervision G.N. and A.D. All authors have read and agreed to the published version of the manuscript.

**Funding:** The study was supported by Foundation for support and development in the field of pediatric hematology, oncology and immunology "Science for Children".

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

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