Copyright © 2019 Wolters Kluwer Health, Inc. All rights reserved.

Luis C. Lopes-Junior, PhD

Denise S. C. Silveira, PhD

Karin Olson, PhD

Emiliana O. Bomfim, MSc

Luciana C. Veronez, PhD

Jéssica C. Santos, PhD

Jonas B. Alonso, BSc

Lucila C. Nascimento, PhD

Gabriela Pereira-da-Silva, PhD

Regina A. G. Lima, PhD

Clown Intervention on Psychological Stress and Fatigue in Pediatric Patients With Cancer Undergoing Chemotherapy

K E Y W O R D S

Psychological stress Cancer-related fatigue Cortisol !-Amylase Pediatric patients with cancer Clown intervention

Background: Clown intervention has been shown to enhance emotional and behavioral processes, but few studies have comprehensively examined the effectiveness of this practice using biomarkers. Objective: The aim of this study was to evaluate the effect of a clown intervention on the levels of psychological stress and cancer-related fatigue in pediatric patients with cancer undergoing chemotherapy. Methods: Sixteen patients who met all criteria from a pediatric oncology inpatient unit in a Brazilian comprehensive cancer care hospital participated in this quasi-experimental study. Eight saliva samples were collected, comprising 4 at baseline and 4 after clown intervention (+1, +4, +9, and +13 hours after awakening). Salivary cortisol and !-amylase levels were determined using high-sensitivity enzyme-linked immunosorbent assay kits. Stress and fatigue were measured by the Child Stress Scale-ESI and the PedsQL Multidimensional Fatigue Scale, respectively. Relationships among stress, fatigue, and biomarker levels were investigated using nonparametric statistics. Results: In comparison with baseline measurements, the total psychological stress and fatigue levels improved after the clown intervention at the

Author Affiliations: Department of Maternal-Infant Nursing and Public Health, University of São Paulo at Ribeirão Preto College of Nursing–PAHO/ WHO Collaborating Centre for Nursing Research Development, Ribeirão Preto, São Paulo, Brazil (Drs Lopes-Júnior, Nascimento, Lima, and Pereira-da-Silva and Mr Alonso); Department of Biochemistry and Immunology, University of São Paulo at Ribeirão Preto Medical School, Graduate Program in Basic and Applied Immunology of the University of São Paulo at Ribeirão Preto Medical School, Ribeirão Preto, São Paulo, Brazil (Drs Silveira, Veronez, and Santos); Edmonton Clinic Health Academy, Faculty of Nursing, University of Alberta, Edmonton, Alberta, Canada (Dr Olson); and Department of Community Health and

Epidemiology, University of Saskatchewan, College of Medicine, Saskatoon, Saskatchewan, Canada (Ms Bomfim).

The authors have no funding or conflicts of interest to disclose. Correspondence: Luis C. Lopes-Junior, RN, OCN, PhD, Department of

Maternal-Infant Nursing and Public Health, University of São Paulo at Ribeirão Preto College of Nursing–PAHO/WHO Collaborating Centre for Nursing Research Development, Avenida dos Bandeirantes, 3900, Campus Universitário, Ribeirão Preto, SP, Brazil, 14040-902 (lopesjr.lc@gmail.com).

Accepted for publication October 24, 2018. DOI: 10.1097/NCC.0000000000000690

Clown Intervention for Pediatric Cancer Patients Cancer NursingW, Vol. 00, No. 0, 2019!1 Copyright © 2019 Wolters Kluwer Health, Inc. Unauthorized reproduction of this article is prohibited.

collection time point +4 hours (P = .003 and P = .04, respectively). Salivary cortisol showed a significant decrease after clown intervention at the collection time points +1, +9, and +13 hours (P < .05); however, !-amylase levels remained unchanged. Conclusion: These findings provide preliminary evidence that clown intervention merits further study as a way to reduce stress and fatigue in pediatric cancer inpatients, and that self-report and biomarker measures are feasible to collect in this patient group. Implications for Practice: Clown intervention as a nonpharmacological intervention may improve stress and fatigue levels in pediatric inpatients with cancer undergoing chemotherapy. n Background Although survival rates for most childhood cancers are higher than ever before, the course of cancer treatment, particularly che- motherapy, still constitutes a stressful and threatening experience for children and adolescents.1–4 Besides suppressing important facets of the immune response such as the activity of NK-cells and the proliferation of T-cells,5,6 cancer treatment and the hos- pitalization process itself often have a psychological impact on pediatric patients with cancer. The complexity of the psycholog- ical stress experienced by pediatric cancer inpatients can exceed their coping strategies, leading to an impaired quality of life, mood disturbances, anxiety, fatigue, and depression.4,7 Cancer-related fatigue (CRF), a subjective and multidimensional phenomenon,8,9 is the most distressing and prevalent treatment- related symptom experienced by pediatric patients with cancer from diagnosis through after the completion of therapy.3,10,11 This especially applies to patients who were treated by chemo- therapy.8,9,11 Frequently and with variable dimensions by the course of the disease, CRF causes a significant negative impact on the child's functional and psychosocial capacity, reducing the quality of life of patients.12 Stress is thought to play an important role in many disorders characterized by fatigue. Stress has been conceptualized as an acute trigger for the onset of fatigue and associated symptoms.12 Despite links between stress and fatigue in other contexts, surprisingly few researchers have examined the association between stress and fatigue in pediatric oncology.13,14 Notably, so far, research for understanding the link between psychological stress and CRF in pediatric inpatients with cancer has not yet been addressed. Circadian deregulation affects the secretion of stress-associated hormones through the psychoneuroimmune-endocrine pathways, which mayhelptoexplainthe associations betweenstress andcancer.15 The few studies that have explored the biological effects of nonpharmacological interventions have not addressed the circa- dian rhythm of the biological markers investigated. The Middle Range Theory of Unpleasant Symptoms and the Psy- choneuroimmunology Framework (PNI) provide the lens through which our research problem was examined. The Theory of Unpleas- ant Symptoms offers a model regarding the experience of and relationships between concurrent symptoms. The 3 major com- ponents of this theory are symptoms, consequences of the symp- tom experience, and influencing factors.16–19 The PNI framework20 indicates possible relationships between behavioral factors and the progression of immunologically-mediated illnesses. According to the PNI, inflammatory and other innate immune mediators can signal back to the central nervous system (CNS), stimulate the production of cytokines, change neuronal function, and cause sickness behaviors and unpleasant symptoms as an adaptive re- sponse.20,21 In the context of our conceptual schema, the PNI supports the idea that immune-to-brain communication cascades might be related to cancer development and treatment-related symptoms such as fatigue, depression, cognitive dysfunction, and sleep disturbances.21 Studies have shown that stress is associated with different illnesses, including cancer, playing an immunosuppressive effect.5 Stress ini- tiates a cascade of information-processing pathways in the CNS and in the periphery, which activates the autonomic nervous system (ANS) or the hypothalamic-pituitary-adrenal (HPA) axis. Cognitive and emotional feedbacks from the cortical and limbic areas of the brain modulate the activity of hypothalamic and brain stem structures directly controlling ANS and HPA activity. Individual differences in the perception and evaluation of external events (appraisal and coping processes) create variability in ANS and HPA activity levels. Autonomic nervous system responses to stress are mediated primarily by the activation of the sympathetic ner- vous system, and subsequent release of catecholamines (mainly norepinephrine and epinephrine) from sympathetic neurons and the adrenal medulla. Hypothalamic-pituitary-adrenal responses are mediated by the hypothalamic production of corticotrophin- releasing factor and arginine vasopressin, which activates the secre- tion of pituitary hormones such as adrenocorticotrophic hormone (ACTH), encephalin, as well as endorphins. Adrenocorticotrophic hormone induces the downstream release of glucocorticoids such as cortisol from the adrenal cortex.20–24 In addition, the normal balance of the HPA axis can be triggered to release cortisol that, in turn, provides feedback to the CNS to stop the hormonal acti- vation of the HPA axis and, thus, homeostasis returns. With pro- longed stress, there is a sustained release of cortisol, which continues to trigger the HPA axis, ultimately leading to inhibition of immune function.5,24 In summary, the HPA axis functions through a nega- tive feedback system: Increased cortisol and other glucocorticoid levels inhibit release of corticotropin-releasing hormone and ACTH from the neurons of the hypothalamus and pituitary gland, respec- tively, leading to a reduction in glucocorticoid levels. A chronic or repeated exposure to a stressor decreases corticotropin-releasing hor- mone, ACTH, and glucocorticoids levels, indicating a reduction in 2!Cancer NursingW, Vol. 00, No. 0, 2019 Lopes-Júnior et al Copyright © 2019 Wolters Kluwer Health, Inc. Unauthorized reproduction of this article is prohibited. negative feedback in the HPA axis, thereby preventing a return to ho- meostasis. In patients with cancer, such dysregulation of the HPA axis and the sympathetic nervous system may be related to the develop- ment, maintenance, and recurrence of cancer.5,22–24 Glucocorticoids control growth, metabolism, and immune function and have a pivotal role in regulating basal function and stress reactivity across a wide variety of organ systems. Most physiologic systems are negatively affected by prolonged exposure to glucocorticoids and catecholamines. It is widely accepted that over time, chronic changes in stress hormones contribute to tumor initiation and development through modulation of the activity of multiple com- ponents of the tumor microenvironment, including DNA repair, oncogene expression by viruses and somatic cells, and production of growth factors and other regulators of cell growth. Once tumor development has begun, neuroendocrine factors can also regulate the activity of proteases, angiogenic factors, chemokines, and adhe- sion molecules involved in invasion, metastasis, and other aspects of tumor progression.20,23–25 In addition, stress hormones, specifically glucocorticoids, influence the polarization of naive Th0 cells into Th1 and Th2 cells. These molecules act on lymphocytes to induce the production of Th2 cytokines and to decrease the production of Th1 cytokine pre- cursors and, consequently, Th1 cytokines.26–29 In physiological concentrations, glucocorticoids may cause a shift from Th1 to Th2 immune responses via alteration of cytokine production. Indeed, patients with cancer who underwent chemotherapy have shown lowered T-cell-mediated secretion of Th1 in comparison with Th2 cytokines.30 The relationship between glucocorticoids and Th1/Th2 cytokine production and differentiation adds an im- portant layer to the relationship of psychological stress and cyto- kines.24,27 Chronic activation of the stress response can lead to increased tumor growth mainly because of the disruption of the deli- cate balance among the CNS, endocrine, and immune systems.5,22–25 With the increase in cancer rates, it is crucial for healthcare pro- fessionals to develop and/or be aware of interventions to relieve the burden of cancer treatment during hospitalizations.1 Pediat- ric cancer inpatients can take advantage of nonpharmacological interventions for managing fatigue and psychological stress.31,32 Within this context, clown intervention stands out as a potential nonpharmacological approach to address these symptoms. It has been shown that this intervention can enhance emotional and behavioral processes, for instance, improving well-being and self-confidence, and reducing stress and anxiety levels.33–35 In ad- dition, evidence suggests that clowns help pediatric patients to better adapt to their hospital surroundings and can distract from and demystify painful or frightening procedures through “doses of fun” to complement traditional clinical interventions.35–37 Positive changes in emotional responses arising from humor and laughter have been correlated with increased pain thresholds and immunity, inversely correlated with stress hormone levels, and linked to positive health.34,38 Our research is based on the assumption that psychological fac- tors may regulate the immune and endocrine response and that patient well-being can significantly affect recovery and response to cancer treatment.20,39 We aimed to evaluate the impact of a clown intervention on the levels of psychological stress and CRF in pediatric patients with cancer undergoing chemotherapy. n Methods Study Design and Ethical Considerations A quasi-experimental study was undertaken from August 2015 to September 2016 at the pediatric oncology inpatient unit in a com- prehensive cancer care in Brazil. We evaluated the effect of a clown intervention on psychological stress and CRF levels. Ethical approval was granted through the Institutional Review Board at Ribeirão Preto College of Nursing-USP, Brazil (815.213/ 2014). Written informed consent was obtained from the parents, and assent of the children was collected for those 6 years or older. Participants We recruited 78 eligible children and adolescents with cancer consecutively at the Pediatric Oncology ward of the Ribeirão Preto Medical School Hospital at University of São Paulo (HCFMRP/ USP). Of the patients invited to be part of the study, 16 met all eligibility criteria and completed the study. Inclusion criteria were as follows: had a diagnosis of pediatric neoplasia; either gender between 6 and 14 years old (because the instruments to assessing stress and fatigue are validated for this population in this age group in Brazil); receiving chemotherapy at the hospital at all points of sample collection; had to be awake, aware, and willing to participate in the study; did not have severe cog- nitive or communication impairments and able to understand the studydesignaswellasanswerthescales;andwerePortuguese speaking. Exclusion criteria were pediatric oncology patients with other chronic comorbidities, autoimmune diseases, or somatic and/or mental illnesses; receiving radiotherapy; receiving end-of-life care; usingantidepressants and/or mood-alteringdrugs;with coulrophobia; with active infectious conditions; in the immediate postoperative period; and with oral lesions and/or dental cavities that could affect saliva collection. The exclusion criteria were restrictive because such situations could affect the circadian concentrations of the biomarkers secondary to the chemotherapy.40,41 Experimental Procedure All patients served as their own controls before and after the inter- vention over a 3-day period. The experimental scheme is shown in Figure 1. Each participant received 1 session of the clown inter- vention and provided a total of 8 saliva samples over a 3-day period (4 samples collected at baseline/preintervention and 4 samples at postintervention). All saliva samples were collected each day at the same time for all participants to maintain comparability among participants and to avoid that differences would be a result of nor- mal daily fluctuations in biomarkers. Preintervention and postintervention samples were collected at +1, +4, +9, and +13 hours after awakening (8:30 AM), that is, at 9:30 AM, 12:30 PM, 5:30 PM, and 9:30 PM, respectively. The 8 points chosen for collection of saliva were based on previous protocols and international recommendations for children and adolescents with chronic conditions, to allow a better cha- racterization of the circadian activity of these hormones.40,42–44 To minimize external influences of clinical procedures on the Clown Intervention for Pediatric Cancer Patients Cancer NursingW, Vol. 00, No. 0, 2019!3 Copyright © 2019 Wolters Kluwer Health, Inc. Unauthorized reproduction of this article is prohibited. measurements, at all sample collection time points, pediatric cancer inpatients had no invasive procedures or any other acute stresses in the last hour before sample collection and underwent preparation for saliva collection. The preparation for saliva collection consisted of not ingesting any food or drinks 1 hour before the procedure and not brushing the teeth or using mouthwash before collection. After preparation had been completed, participants were requested to refrain from swal- lowing briefly (for 30 seconds) and then “drooling” the saliva from the mouth as much as possible directly into the collection device. SALIVARY CORTISOL AND !-AMYLASE Cortisol is the main endogenous glucocorticoid capable of influenc- ing the retro inhibition of the HPA axis45,46 and is present in the free form in saliva. Salivary cortisol levels reflect the activity of the HPA axis41 and can be used to assess stressor responses or to de- termine the efficacy of interventions intended to reduce stress.45 Salivary cortisol as a biological stress marker emerged in pedi- atric research as an easy-to-collect and relatively cheap method, especially to prevent stress induced by the blood collection proce- dure, which would compromise the reliability of the results.41,46 Saliva has been considered a noninvasive approach to assess a range of biological substances, including cortisol, immunoglobulin A, !- amylase (sAA), cytokines, among other biomarkers.45 In addition, salivary measures are being used on a large scale in biobehavioral and clinical research to understand the impact of stress on health and disease progress.42,45 Salivary cortisol levels are statistically reliable measures of the free fraction in the child-juvenile population.46 The circadian rhythm of cortisol is known, floating during the day according to a pre- dictable cycle and demonstrating variations. Cortisol levels tend to peak about 20 to 30 minutes after awakening, drop by half in the midafternoon, and are at their lowest around midnight.41,47 An estimated gap of 15 to 30 minutes exists between the stressor and the increased production and release of plasma cortisol and an additional 2-minute gap before increased cortisol is detected in the saliva.47 For intervention studies, the number of samples should include at least 2 measures: the baseline cortisol levels and the postintervention measure. The baseline samples present a measure of the normal cortisol levels of the child and adolescent on a typical day of hospitalization. Another biomarker that has been reported in the scientific literature to reliably reflect changes related to psychological stress is sAA43,48,49 (1,4-!-glucan-4-glucanohydrolase, EC 3.2.1.1.), one of the most important enzymes in the saliva and produced by the acinar cells of the salivary glands.50–52 Its release is provoked by the activation of the ANS through specific neurotransmitters and bioactive peptides (eg, acetylcholine, vasoactive intestinal pep- tide, and P substance), providing an adequate measure of the sym- pathetic activities and exhibiting a circadian rhythm opposite to that of cortisol.44,48–50 Corticosteroids can cause the suppression of the HPA axis40 and change cortisol levels. Many childhood cancer treatment protocols include high doses of corticosteroids. Conse- quently, the use of cortisol as a stress measure may not be sensitive to small changes in the concentrations of this biomarker in pediat- ric patients with cancer.40 A promising alternative to cortisol is sAA, which is not affected by the use of corticosteroids.41,53 Intervention Clown intervention was performed by undergraduate and gradu- ate students from the USP who are members of the Companhia do Riso (The Laugh Company), which is an outreach project aimed to lift children/adolescents moods during hospitalization and those of their families and staff.54 Children, accompanied by their parents, interacted simultaneously with 2 clown volunteers in the pediatric oncology ward for approximately 30 minutes. Clowns performed interactive activities while adapting their techniques to each patient's age and psychological condition and thus varied by child. Overall, clowns delivered play sessions for patients using improvisation and various methods for entertaining the children (eg, magic tricks, puppets, songs, etc). All clowns attended specific training and orientation sessions focused on practical work situations to de- velop theatrical and artistic/clown competences in addition to social, psychological, and pedagogical skills.54 Because this study is not a standalone research and took place in a public hospital where the intervention is part of the routine service since 1995, Figure 1. Experimental scheme. 4!Cancer NursingW, Vol. 00, No. 0, 2019 Lopes-Júnior et al Copyright © 2019 Wolters Kluwer Health, Inc. Unauthorized reproduction of this article is prohibited. there were no activities to increase clown compliance or adher- ence. Given its long history of availability to all children as part of standard care, we chose not to randomly assign patients to not receive this care. Measures The Child Stress Scale (Escala de Estresse Infantil, ESI) is derived from the Child Stress Symptoms Inventory and was validated in the Portuguese language in the Brazilian population by 2 psychologists.55 The ESI is used to evaluate stress in children aged 6 to 14 years and includes 4 dimensions (physical, psy- chological, psychological with depressive components, and psychophysiological stress). The answers to the 35 items are given using a 4-point Likert scale. The scoring procedure is based on cutoff points for each of the 4 dimensions, with higher scores indicating higher stress levels. A blinded psychologist performed the scoring. Previous internal consistency estimate for this age group in Brazil was Cronbach's ! = .90.55 In the present study, the Cronbach's ! was high (! = .86). We administered the ESI on day 1 (baseline) between 12:30 PM and 5:30 PM (+4 and +9 hours after awakening) and on day 3 (postintervention) between 9:30 AM and 12:30 PM (+1 and +4 hours after awakening). The Pediatric Quality of Life Inventory (PedsQL) Multidi- mensional Fatigue Scale (MFS) includes 3 dimensions that use a 5-point Likert interval scale: general fatigue, fatigue and sleep/ rest, and cognitive fatigue. For scoring, items are reversed scored and linearly transformed to a scale of 0 to 100; higher scores indi- cate lower fatigue levels.56 In Brazil, this instrument has been val- idated for the pediatric oncology population with 8 years or older, and its reliability as measured by the internal consistency of items and dimensions was acceptable, with Cronbach's ! between .70 and .90 for both self and proxy versions.57 We administered both the PedsQL MFS self-report and proxy (parents' report) versions on the same days and times as the ESI. Self-report and proxy reports Cronbach's ! coefficients were ! = .72 and ! = .88, re- spectively, in this study. Laboratory Methods Saliva samples (2 mL) were collected from each participant using the passive drool technique. Participants were instructed to drool through a sterile saliva collection aid (Salimetrics, LCC, State College, Pennsylvania) to collect saliva directly into a cryovial, which retains saliva contaminants. After saliva collec- tion, the samples were taken to the Laboratory of Genomics and Immunobiology. Saliva samples were run in duplicates (50 "L each) along with standards and controls in all assays. !-Amylase activity was measured as described previously.58 Free cortisol levels in saliva were measured in duplicates using a commercially available chemiluminescence assay kit. Interassay and intra-assay variation was less than 10%. In addition, both salivary cortisol and sAA concentrations were measured using high-sensitivity enzyme-linked immunosorbent assay kits for human samples following the manufacturers' instructions (Salimetrics LLC). Statistical Analysis Statistical analyses were performed using R 3.2.3 Wooden Christmas Tree for Windows (R Development Core Team, 2016) and GraphPad Prism version 6.0 (GraphPad Software, Inc, San Diego, California). Descriptive analyses were con- ducted to characterize the samples. Bivariate association anal- yses were performed to compare psychological and fatigue stress scores and biomarker concentrations before and after the intervention using the nonparametric paired Wilcoxon signed rank test. Also, Spearman correlation analysis was used to examine putative relationships of saliva measurements with subjective ratings from the stress and fatigue scoring and bio- markers levels. In addition, the area under the curve (AUC), which is a measure of the total daily production of a biomarker based on the log-trapezoidal method,59 was calculated for analysis of biomarker data. A 2-tailed P value < .05 was consid- ered to be statistically significant. n Results Sample Characteristics During the recruitment period, 78 pediatric patients with cancer were eligible (37 boys/41 girls) for this study. Of those, 16 (9 boys/7 girls) consented to participate (Figure 2). The mean (SD) age of the patients was 11.4 (3.4) years, 50% were white, and 68.7% had completed primary school. The most prevalent tumor was osteosarcoma (n = 6), followed by leukemia (n = 4) and lymphoma (n = 4). Most (81.3%, n = 13) were pri- mary neoplasia, and among the 16 patients, 68.7% (n = 11) had metastases. In addition, 56.3% (n = 9) were using corticosteroids during the chemotherapy protocol (Table 1). Salivary Cortisol and sAA Trajectories After the Clown Intervention Salivary cortisol showed significant decreases between preintervention (baseline) and postintervention values at the collection time points + 1, +9, and +13 hours (P < .05) (Figure 3A). The AUC for salivary cortisol showed no significant difference between baseline and postintervention (cortisol AUC baseline/postintervention: 0.53 [0.36] vs 0.47 [0.36] "g/dL, P = .790). The clown intervention had no significant effect on salivary sAA levels between baseline and postintervention measurements (P > .05) (Figure 3B). Similarly, no significant difference was detected for sAA AUC (sAA AUC baseline/postintervention: 241 [109.3] vs 241 [129.3] U/mL, P = .755) (Figure 3B).

Psychological Stress and Fatigue Symptoms at Baseline and Postintervention

The overall psychological stress score decreased in the postinter- vention compared to baseline (baseline/postintervention: 32.1 [17.6] vs 19.6 [14.1], P = .003), indicating lower stress levels after the clown intervention. The 3 domains of the ESI, that is,

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psychological reactions (P = .04), psychological reactions with depressive component (P = .04), and psychophysiological reac- tions (P = .006), decreased after the clown intervention (Table 2).

The overall PedsQL MFS scores changed significantly by pa- tient self-report from baseline to postintervention (66.3 [19.6] vs 81.5 [17.3], P = .04), indicating lower levels of fatigue after the intervention. However, when the domain scores were analyzed separately, there were no statistically significant differences. Fa- tigue levels from parent proxy reports were unchanged after the clown intervention (P = .379).

Correlation Between Stress/Fatigue Dimensions and Salivary Cortisol AUC/ sAA AUC

The total ESI stress scores from the pediatric patients correlated positively with AUC for cortisol at baseline (r = 0.35, P = .03), indicating that higher baseline stress scores positively correlated with increased levels of cortisol at preintervention. Decreased stress scoring after the clown intervention correlated positively with decreased levels of cortisol (r = 0.02, P = .04). Conversely, the total ESI stress scores from the patients correlated negatively with AUC for sAA at baseline (r = !0.57, P = .02), but not at the postintervention (Table 3). Regarding the total PedsQL fatigue

score, we have found no correlations between cortisol AUC and sAA AUC neither baseline nor post-intervention (Table 3).

n Discussion

This study was conducted to investigate the impact of a clown intervention as a nonpharmacological intervention on stress and fatigue levels in pediatric patients with cancer undergoing chemotherapy, specifically whether a clown intervention im- proved psychological stress and CRF as well as altered salivary cortisol and sAA. Overall, the total psychological stress scoring for pediatric patients with cancer as well as their fatigue levels improved after the clown intervention (P = .003 and P = .04, respectively). Salivary cortisol showed a significant decrease after the intervention at the collection time points +1, +9, and +13 hours (P < .05); however, SAA levels remained unchanged. To the best of our knowledge, this is the first study to investigate the impact of the clown intervention on cortisol and sAA and to associate them with CRF and stress in pediatric patients with cancer undergoing chemotherapy. Abnormal cortisol release in patients with cancer may con- tribute to chronic inflammation,60,61 promote tumor development and growth, and consequently can affect the response to treatment.4 In our study, salivary cortisol levels decreased (at +1, +9, and +13 hours after awakening) upon clown intervention. These Figure 2. Patient recruitment flowchart. 6!Cancer NursingW, Vol. 00, No. 0, 2019 Lopes-Júnior et al Copyright © 2019 Wolters Kluwer Health, Inc. Unauthorized reproduction of this article is prohibited. findings are in line with recent reports that have shown lower cortisol levels after clown intervention in pediatric patients with acute disorders.62,63 No correlation was detected between cortisol AUC and sAA AUC at baseline (r = !0.20, P = .437) or postintervention (r = !0.42, P = .132). This result is consistent with previous studies.43,44,49,64 Indeed, this endorses sAA activity as a measure clearly distinct from cortisol, once sAA and cortisol outputs representing different systems, that is, the ANS axis and the HPA axis, respectively. The daily pattern of sAA and cortisol activity in children parallels pre- vious findings as well, such that in adults, the trajectory of sAA activity over the course of a day was opposite of that observed for cortisol, with lowest levels 1 hour after awakening and in- creasing levels over the day.49,60 Our results showed a decrease in total stress scores and in the 3 domains in pediatric cancer inpatients after the clown interven- tion, as assessed by the ESI. These results are consistent with the 1 study that evaluated the impact of a humor therapy program on stress levels in pediatric inpatients with different diagnoses and showed that the children in the intervention group presented lower scores on the Parker test—a validated scale for evaluating emotional stress, than children in the nonintervention group.63 We have also found that total the fatigue scores of our partici- pants, as assessed by the PedsQL MFS (self-version), significantly improved after the clown intervention (P = .04). This finding is in line with 1 study that reported that hospitalized children and adolescents who received clown intervention had an increase in self-reported psychological well-being as well as in emotional re- sponses, compared with those in the control group.65 Contrary to these results, 1 recent pilot study that examined the feasibility of longitudinal testing of psychophysiological parameters of stress and fatigue in pediatric osteosarcoma patients hospitalized for chemother- apy submitted to clown intervention have shown that the outcomes measured by PedsQL MFS (self-report and parents-report) were not changed after clown intervention.66 Probably, these different results may be partly justified by the small sample size from the pilot study. n Limitations The sample size was small, which limits the generalizability of findings. Future studies with larger sample sizes are needed to validate the findings. Because data collection was at a single pediatric oncology unit in Brazil and because of the rigid Figure 3. (A) Salivary !-amylase and (B) salivary cortisol trajectories in pediatric cancer inpatients upon clown intervention (n = 16). The graphs show the mean and standard deviation of biomarkers trajectories of the pediatric patients with cancer at preintervention (baseline) and at postintervention. +1, +4, +9, and +13 hours after awakening (8:30 AM), that is, at 9:30 AM, 12:30 PM, 5:30 PM, and 9:30 PM, respectively. * P < .05 by the Wilcoxon signed rank test. Table 1 • Sociodemographic and Clinical Characteristics of Pediatric Inpatients With Cancer (n = 16) N (%) Mean (SD), Range Sociodemographic characteristics Gender (%) Male 9 (56.3) Female 7 (43.7) Age (years) 11.4 (3.44), 6–14 Ethnicity White 8 (50) Black 8 (50) Education Preschool 1 (6.3) Primary school 11 (68.7) Secondary school 4 (25) Clinical characteristic Body weight. kg 40.1 (13.9), 18.8–60 BMI. kg/m2 18 (3.7), 13–26.3 Body surface area, m2 1.2 (0.3), 0.7–1.65 Diagnoses (%) Osteosarcoma 6 (37.4) Leukemia 4 (25) Lymphoma 4 (25) Wilms tumor 1 (6.3) Ependymoma 1 (6.3) Metastases No 11 (68.7) Yes 5 (31.3) Previous treatments Surgery 10 (62.5) Radiation 0 (0) Chemotherapy 0 (0) Surgery and radiation 2 (12.5) No previous treatment 4 (25) Corticosteroid use No 9 (56.3) Yes 7 (43.7) Abbreviation: BMI, body mass index. Clown Intervention for Pediatric Cancer Patients Cancer NursingW, Vol. 00, No. 0, 2019!7 Copyright © 2019 Wolters Kluwer Health, Inc. Unauthorized reproduction of this article is prohibited. inclusion and exclusion criteria, the number of participants was restricted and consequently small. A quasi-experimental design was used instead of a randomized controlled trial, mainly because of the nature of the intervention where the study was undertaken (standard of care). Observed changes are difficult to interpret be- cause some reported changes might simply reflect an effect of re- peated measurement; therefore, the results should be viewed with caution. Although the stress hormones were measured at several times, psychological data were available only at baseline and post- intervention. Thus, it remains unknown whether the intervention produced only short-term effects on perceived stress and fatigue or whether the effect of the intervention persisted for some hours. Finally, the heterogeneity of pediatric tumors included in the study may have affected the results concerning the biomarker trajectories. In addition, we cannot exclude that the type of procedure, timing and type of treatment, time of the next treatment/procedure, stage/ grade of tumor, presence of metastases, and prognosis of the pe- diatric patients with cancer may also have influenced our results. n Clinical Implications for Nursing Despite these limitations, this novel study is valuable in its efforts to address gaps in the literature with regard to the impact of the clown intervention in pediatric patients with cancer undergoing chemotherapy. In summary, our results show that clown interven- tion had beneficial effects for reducing psychological stress scores as well as cortisol levels and CRF scoring in pediatric cancer inpa- tients. It is important to highlight that none of the children and adolescentswhodeclinedtoparticipatein our studyhadcoulrophobia. However, if that was the case, other types of nonpharmacological intervention such as programmed physical exercises to manage stress, as well fatigue in those patients, especially because exercise interventions for CRF are feasible and safe, would be recom- mended.31,32 Our findings also support the continued investigation of nonpharmacological interventions for management and im- provement of health outcomes during hospitalization in pediatric oncology.31,32,67 This study provides scientific evidence that healthcare profes- sionals in pediatric oncology could use to minimize the psy- chological burden of hospitalizations. There is great potential to advance cancer research by evaluating psychoneurological symp- tom clusters via nonpharmacologic intervention and their inter- actions with biological pathways and developmental changes in symptom severity over time.68–70 Conclusion Our findings provide preliminary evidence that the clown inter- vention merits further study as a way to reduce stress as well as fatigue in pediatric cancer inpatients, as patient and proxy reports and biomarker measures of stress and fatigue are feasible to col- lect in this group. Our results suggest that the clown intervention as a nonpharmacological intervention may improve stress and fatigue levels in pediatric patients with cancer undergoing chemotherapy. Future research should focus on a specific tumor type, have homogenous samples, and use a more detailed investigation with robust statistical analyzes. Importantly, future studies could also identify pediatric cancer inpatient profiles most likely to benefit from this type of intervention, in terms of age, gender, frequency of clown visits, and follow-up period. Table 2 • Scoring of Fatigue and Stress Dimensions in Pediatric Inpatients With Cancer (n = 16) at Baseline and Postintervention Instrument Baseline, Mean (SD)a Postintervention, Mean (SD)a P PedsQL dimensions (self-report) General fatigue 69 (19.3) 77.7 (21) .18 Sleep/rest fatigue 60.1 (22.8) 66.6 (18.4) .32 Cognitive fatigue 69.9 (25.6) 77.7 (21) .20 Total PedsQL fatigue score 66.3 (19.6) 81.5 (17.3) .04b PedsQL dimensions (parents' report) General fatigue 66.9 (17.4) 66.9 (23.7) .32 Sleep/rest fatigue 51.7 (22.3) 57.1 (22.9) .52 Cognitive fatigue 81.2 (19.2) 82.7 (21.9) .52 Total PedsQL fatigue score 66.1 (17) 68.4 (17) .37 ESI domains Physical reactions 6.7 (5.5) 4.4 (4.2) .07 Psychological reactions 10.6 (5.9) 7 (6.1) .04b Psychological reactions with depressive component 6.4 (4.5) 4.2 (4) .04b Psychophysiological reactions 8.2 (4.6) 4 (2.6) .006b Total ESI™ score 32.1 (17.6) 19.6 (14.1) .003b Abbreviations: ESI, Child Stress Scale; PedsQL, Pediatric Quality of Life Inventory Multidimensional Fatigue Scale. Bold data indicates statistically significant (P < .05). aValues given are mean (SD). bP < .05 (postintervention values that significantly differ from baseline). Table 3 • Spearman Correlation Coefficients of Salivary Cortisol and sAA and ESI and PedsQL Scoring of Pediatric Inpatients with Cancer (n = 16) at Preintervention and Postintervention Cortisol AUC sAA AUC Pre (!)a P Post (!)a P Pre- (!)a P Post (!)a P Total ESI stress score 0.35 .03 0.02 .04 !0.57 .02b !0.27 .34 Total PedsQL fatigue score !0.27 .30 !0.26 .36 0.44 .08 0.37 .18 Abbreviations: AUC, area under curve; ESI, Child Stress Scale; PedsQL, Pediatric Quality of Life Inventory Multidimensional Fatigue Scale; sAA, !-amylase. Bold data indicates statistically significant (P < .05). aValues given are mean. bP < .05. 8!Cancer NursingW, Vol. 00, No. 0, 2019 Lopes-Júnior et al Copyright © 2019 Wolters Kluwer Health, Inc. Unauthorized reproduction of this article is prohibited. 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