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BACKGROUNDPaediatric neuroblastoma and brain tumours account for a third of all childhood cancer-related mortality. High-risk neuroblastoma is highly aggressive and survival is poor despite intensive multi-modal therapies with significant toxicity. Novel therapies are desperately needed. The Zika virus (ZIKV) is neurotropic and there is growing interest in employing ZIKV as a potential therapy against paediatric nervous system tumours, including neuroblastoma.METHODSHere, we perform an extensive meta-analysis of ZIKV infection studies to identify molecular mechanisms that may govern the oncolytic response in neuroblastoma cells. We summarise the neuroblastoma cell lines and ZIKV strains utilised and re-evaluate the infection data to deduce the susceptibility of neuroblastoma to the ZIKV oncolytic response. Integrating transcriptomics, interaction proteomics, dependency factor and compound datasets we show the involvement of multiple host systems during ZIKV infection.RESULTSWe identify that most paediatric neuroblastoma cell lines are highly susceptible to ZIKV infection and that the PRVABC59 ZIKV strain is the most promising candidate for neuroblastoma oncolytic virotherapy. ZIKV induces TNF signalling, lipid metabolism, the Unfolded Protein Response (UPR), and downregulates cell cycle and DNA replication processes. ZIKV is dependent on SREBP-regulated lipid metabolism and three protein complexes; V-ATPase, ER Membrane Protein Complex (EMC) and mammalian translocon. We propose ZIKV nonstructural protein 4B (NS4B) as a likely mediator of ZIKVs interaction with IRE1-mediated UPR, lipid metabolism and mammalian translocon.CONCLUSIONSOur work provides a significant understanding of ZIKV infection in neuroblastoma cells, which will facilitate the progression of ZIKV-based oncolytic virotherapy through pre-clinical research and clinical trials.KEYPOINTSThe Zika virus may provide the basis for an oncolytic virotherapy against NeuroblastomaMost paediatric neuroblastoma cell lines are susceptible to Zika viral infectionWe identified molecular mechanisms that may induce the oncolytic response in NeuroblastomaContribution to the fieldThe ability to both induce direct oncolysis and provoke an anti-tumoral immune response makes oncolytic virotherapy an attractive candidate to combat aggressive and heterogenous cancers, such as high-risk neuroblastoma. To progress oncolytic virotherapy to clinical trial it is essential to understand the host mechanisms the virus manipulates to kill cancer cells, alongside any pathology as a consequence of infection of normal cells. Here, we show that ZIKV efficiently infects and induces oncolysis of paediatric neuroblastoma cells and propose a potential TNF pathway-driven immune response. ZIKV’s specificity for infection of nervous system cancer cells, while rarely causing nervous system-related pathology in young children, addresses many of its safety concerns. The inclusion of more effective and less toxic novel therapies, such as a potential ZIKV-based therapeutic, in multimodal treatment regimens will pave the way for improving patient long-term health and overall survival.

Pediatric patients with high-risk neuroblastoma have poor survival rates and urgently need more effective treatment options with less side effects. As novel and improved immunotherapies may fill this need, we dissected the immunoregulatory interactions in neuroblastoma by single-cell RNA-sequencing of 25 tumors (10 pre- and 15 post-chemotherapy, including 5 pairs) to identify strategies for optimizing immunotherapy efficacy. Neuroblastomas were infiltrated by NK, T and B cells, and immunosuppressive myeloid populations. NK cells showed reduced cytotoxicity and T cells had a dysfunctional profile. Interaction analysis revealed a vast immunoregulatory network and identified NECTIN2-TIGIT as a crucial immune checkpoint. Combined blockade of TIGIT and PD-L1 significantly reduced neuroblastoma growth, with complete responses in vivo. Moreover, addition of TIGIT blockade to standard relapse treatment in a chemotherapy-resistant Th-ALKF1174L/MYCN 129/SvJ syngeneic model significantly improved survival. Concluding, our integrative analysis of neuroblastoma’s vast immunoregulatory network provides novel targets and a rationale for immunotherapeutic combination strategies.

10039 Background: Children with relapsed and refractory neuroblastoma (RR-NBL) have poor outcomes. Early identification of children at greatest risk of relapse could mean timelier modifications of treatment to improve outcomes. High levels of adrenergic neuroblastoma mRNAs in blood of children with stage M neuroblastoma receiving frontline treatment predict poor outcome (Viprey PMID: 24590653). Since these markers have not been thoroughly studied in the RR-NBL population, we have prospectively evaluated the prognostic potential of the adrenergic neuroblastoma mRNAs paired-like homeobox 2B (PHOX2B ) and tyrosine hydroxylase (TH) in blood from children with RR-NBL treated in the BEACON-Neuroblastoma trial (NCT02308527). Methods: Blood samples collected at baseline from 88 children were analysed by reverse transcriptase polymerase chain reaction (RTqPCR) for PHOX2B and TH mRNAs. The prognostic power of these mRNAs was evaluated using Kaplan-Meier survival curves and Cox proportional hazards regression. Progression-free (PFS) and overall survival (OS) were calculated from the date that the blood sample was taken at screening to the date of an event; progression, disease recurrence, death or censored alive at the last clinical evaluation. Results: Of the children in this cohort, 58 (66%) had relapsed and 30 (34%) had refractory disease. Twenty-three (26%) had MYC-N amplified tumours. TH and PHOX2B mRNAs were detected in 55% and 60% of blood samples respectively; the correlation coefficient between TH and PHOX2B was 0.75. Higher levels of TH, PHOX2B mRNAs or both combined were associated with reduced PFS and OS (Table). For TH, median PFS for children with TH levels below the median was 12 months (95%CI, 4.6–13 months) versus 5.5 months (95%CI, 1.8–9.4 months) for those children with TH levels above the median. For PHOX2B, median PFS for children with PHOX2B levels below the median was 11.5 months (95%CI, 7.6–34 months), compared to 5.7 months (95%CI, 1.8–10.5 months) where levels were above the median. Conclusions: TH and PHOX2B mRNAs in blood collected at baseline identify children with refractory or relapsed neuroblastoma at greatest risk of progression or death. In the RR-NBL setting, this simple blood test could be used to stratify treatment strategies. Clinical trial information: NCT02308527. [Table: see text]

Neurotoxicity is an off-tumour, on-target side effect of GD2-directed immunotherapy with monoclonal antibodies. Here, we report the frequency, management and outcome of patients enrolled in two prospective clinical trials who experienced severe neurotoxicity during immunotherapy with the anti-GD2 antibody dinutuximab beta (DB) administered as short-term infusion (HR-NBL1/SIOPEN study, randomisation R2, EudraCT 2006-001489-17) or as long-term infusion (HR-NBL1/SIOPEN study, randomisation R4, EudraCT 2006-001489-17 and LTI/SIOPEN study, EudraCT 2009-018077-31), either alone or with subcutaneous interleukin-2 (scIL-2). The total number of patients included in this analysis was 1102. Overall, 44/1102 patients (4.0%) experienced Grade 3/4 neurotoxicities (HR-NBL1 R2, 21/406; HR-NBL1 R4, 8/408; LTI study, 15/288), including 27 patients with severe neurotoxicities (2.5%). Events occurred predominantly in patients receiving combined treatment with DB and scIL-2. Neurotoxicity was treated using dexamethasone, prednisolone, intravenous immunoglobulins and, in two patients, plasmapheresis, which was highly effective. While neurological recovery was observed in 16 of 21 patients with severe neurotoxicities, 5/1102 (0.45%) patients experienced persistent and severe neurological deficits. In conclusion, severe neurotoxicity is most commonly observed in patients receiving DB with scIL-2. Considering the lack of clinical benefit for IL-2 in clinical trials so far, the administration of IL-2 alongside DB is not recommended.

BACKGROUND Malignant paediatric nervous system tumours, such as medulloblastoma, ATRT and high-risk neuroblastoma commonly harbour tumour cells with stem-like features which are highly tumorigenic and resistant to conventional therapies. These tumours can exhibit high lethality and may result in severe sequelae that significantly affect paediatric patients' quality of life. Oncolytic virotherapy exploits viruses that preferentially infect and destroy tumour cells. These viruses present a unique advantage in targeting highly heterogeneous cancers as they possess a secondary mechanism of action, through which they induce an anti-tumoral immune response. The Zika virus (ZIKV) is capable of infecting and destroying aggressive human paediatric brain tumour and neuroblastoma cells in vitro. ZIKV effectively reduces brain tumour size in mice (xenograft model) and canines (naturally occurring) and can induce an immune response against canine brain tumours. METHODS Employing global expression omics profiling of ZIKV infection and mapping of viral protein-host protein interactions, we aim to elucidate the mechanisms which underpin ZIKVs therapeutic properties, both at the molecular and cellular pathway levels. RESULTS Through extensive transcriptome profiling of ZIKV-infected paediatric brain tumour, neuroblastoma and NPCs, we have identified a variety of pathways which are involved in the ZIKV oncolytic response in the tumour cells and its neuro-dysregulation of NPCs. Despite both brain tumour and neuroblastoma cells undergoing ZIKV-induced oncolysis, we observed there to be a heterogeneous response within these different tumour cells at the molecular level to lead to oncolysis. Additionally, the infected tumour cells demonstrate elevated immune system profiles which alludes to the immune response that ZIKV may raise within the patient’s body against the paediatric tumour. Analysing our findings alongside the neuro-dysregulation we observe in our ZIKV-infected NPCs is allowing us to build a safety profile for employing a ZIKV-based therapy, whilst contributing to the growing knowledge of Congenital ZIKV Syndrome.

The UK government has now revised its advice on the safety of eggs for vulnerable groups, such as pregnant women, infants and young children, confirming that they are safe to eat ‘runny’ or even raw, provided they are British Lion eggs. The government's Scientific Advisory Committee on Nutrition (SACN) has also re-confirmed that eggs can be introduced from around 6 months when complementary feeding begins, despite their allergenic potential. Furthermore, it suggests that deliberate exclusion or delays in introducing eggs beyond 6–12 months may increase the risk of egg allergy. This article discusses the revised recommendations in more detail and sets the SACN recommendations in the context of developing research on egg allergy. It compares advice on egg consumption for non-atopic and high-risk infants in the UK, the US and Australia, and examines the evidence on the influence of maternal diet during lactation on the risks of infant egg allergy. Finally, it provides an update on the nutritional benefits of eggs for pregnant women, infants and young children, including new research on critical but lesser known nutrients such as iodine and choline, with consideration of the suitability of eggs in baby-led weaning.

10002 Background: The BEACON phase II trial (NCT02308527) addressed a number of questions in children with relapsed/refractory high-risk neuroblastoma (RR-HRNB). Here we report the chemo-immunotherapy randomisation, assessing if anti-GD2 (dB) demonstrates activity when added to chemotherapy. Methods: Patients aged 1-21 years with RR-HRNB with adequate organ function and performance status were randomised in a 1:2 ratio to receive chemotherapy alone or with dB, given concurrently as a 7 day continuous infusion (10 mg/m2/24hr). As the trial had a factorial design, some patients were also randomised between chemotherapy regimens (temozolomide (T) versus Temozolomide-Topotecan (TTo); this randomisation closed soon after the dB randomisation opened and all patients subsequently received TTo chemotherapy. Cross-over to dB with topotecan and cyclophosphamide was allowed for patients randomised to chemotherapy alone who experienced disease progression. The primary outcome measure was best response (complete or partial) at any point during the first 6 courses of treatment, by RECIST or International Neuroblastoma Response Criteria for patients with measurable and evaluable disease respectively. Progression free and overall survival (PFS & OS) and safety were secondary outcomes. The success criterion for proceeding to a Phase 3 trial was a one-sided p-value (1p) less than 0.23 for Objective Response Rate (ORR). Results: From Aug 2019 to Feb 2021, 65 patients were randomised to chemotherapy alone (3 T, 19 TTo) or with dB (6 dBT, 37 dBTTo). Median age was 4 years; 48 and 17 had measurable and evaluable disease respectively; 29 and 36 had refractory and relapsed disease respectively; 19 had MYCN amplification. Baseline characteristics were balanced between arms. Response was assessable in all patients. The ORR was 18% with chemotherapy alone and 35% for patients receiving chemotherapy with dB (risk ratio 1.66, 80% confidence interval (CI) 0.9 to 3.06, 1p = 0.19). 1-year PFS was 27% for chemotherapy alone, and 57% for those receiving chemotherapy +dB (HR 0.63, 95% CI 0.32 to 1.25, p = 0.19). Twelve patients in the chemotherapy only arm crossed over to receive dB at progression. OS did not differ between the arms: HR = 0.99, 95% CI 0.42 to 2.36, p = 0.99. Nine (41%) patients receiving chemotherapy alone and 13 (30%) receiving chemotherapy plus dB had grade ≥3 toxicities (CTCAE v4.0). Neurotoxicities were more common in patients receiving dB compared to chemotherapy alone (Grade 1-2: 67.4% vs 13.6%, Grade 3: 9.3 vs 0%). Other toxicities were similar with and without dB. Conclusions: The Phase 2 success criterion for ORR was met and PFS is also encouraging. The addition of dB to temozolomide-based chemotherapy shows promising activity in patients with RR-HR-NB. Clinical trial information: NCT02308527.

Objectives A programme was introduced in Canterbury, New Zealand to evaluate the diagnosis and treatment of frenulum releases in newborn infants with suspected tongue-tie (ankyloglossia). The primary goals were to support breastfeeding and ensure that unnecessary surgery was avoided. Methods Local healthcare professionals reached consensus on a pathway for improving management of infants with tongue-tie and breast-feeding difficulties. This embedded an expert breast-feeding review and assessment of lingual function using a validated method, the Bristol Tongue-tie Assessment Tool (BTAT). Infants with breastfeeding problems related to tongue-tie had a frenotomy at a hospital outpatient clinic. An education programme was developed to support introduction of the new clinical pathway and included seminars and online information for healthcare professionals and the general public. Results Frenotomy intervention rate reduced markedly from 11.3% in 2015 to 3.5% by mid-2017. Feeding methods were not different before or after surgery between infants who received a frenotomy and those who did not. Initially, the BTAT threshold for frenotomy was set at ≤5, however the final clinical pathway combined a breastfeeding assessment and a BTAT threshold of ≤4. The education programs assisted with the changes in practice, while increased use of the clinician guidance and public health information websites confirmed growing awareness of tongue-tie and community breastfeeding support. Conclusions Establishing consistent multidisciplinary assessment of tongue-tie in infants with feeding difficulties led to a marked reduction in frenotomy intervention rate. 23% of the frenotomy group in the 2016 audit showed a significant improvement in the ability to breastfeed, but overall there was no difference in the feeding pattern of infants who either received or were declined a frenotomy. The development of a supportive education programme and availability of online information about tongue-tie for health professionals and consumers contributed to successful uptake of the new clinical pathway.

Eggs are a nutrient-dense, relatively inexpensive and convenient food, suitable for consumption by pregnant and breastfeeding women and infants. However, there appears to be some confusion among the public regarding the safety of egg consumption by these groups. Some evidence suggests that delayed introduction of potential food allergens such as eggs during weaning may be counterproductive for allergenic risk, and that introduction of the allergenic protein during a proposed ‘critical window’, at 4–7 months, may be necessary to induce tolerance. This hypothesis is currently being tested by two major studies, while two expert committees (Scientific Advisory Committee on Nutrition and Committee on Toxicity of Chemicals in Food, Consumer Products and the Environment) are also reviewing evidence on complementary feeding and allergic risk, respectively. This article discusses historic and current advice on egg consumption during pregnancy and breastfeeding and in complementary feeding. It examines the evidence base for the advice in the context of both the microbiological safety of eggs and risks of egg allergy in these groups, presents a new analysis of nationally representative infant feeding data and reviews the latest nutritional composition data for British eggs in the context of the maternal and infant diet. It appears that historic concerns about food safety and allergy may have resulted in unnecessary avoidance of eggs during the introduction of complementary feeding.

Sarcopenia is an important health issue for older people. It is closely linked with frailty and malnutrition and can significantly reduce both health and quality of life for those affected. Sarcopenic decline in muscle mass can start as early as the fourth and fifth decade of life, so the maintenance of muscle mass throughout adulthood, through regular physical activity and a balanced diet, should be an important consideration in reducing the risk of sarcopenia in older age. Maintaining regular exercise throughout older age remains key to the treatment of sarcopenia, as does an adequate intake of nutrients, including high-quality protein and vitamin D. A significant proportion of older people fail to meet the recommended requirements for protein; it has also been suggested that the requirements in existing recommendations could be higher. Evidence is emerging that an adequate intake of protein at each meal may be required to optimise muscle synthesis in older people. Eggs are an inexpensive, widely available and easily digestible source of high-quality protein and contain a significant proportion of leucine, an amino acid that is important for muscle synthesis, as well as many other nutrients of significance for older people, including vitamin D and omega-3 fatty acids. For many older people, eggs are a familiar and acceptable protein food at breakfast and other meals. Encouraging both those approaching older age and older people to include eggs more frequently, as part of a healthy, balanced diet and in addition to physical activity, could help them maintain their muscle strength and function, thereby preserving their functional capacity and reducing morbidity, mortality and healthcare costs associated with sarcopenia.

To determine the relative impact of three iso-caloric breakfast meals, of variable composition, on satiety, hunger and subsequent intake of energy.In a three-way, crossover design, 30 healthy men (age of 21.7 ± 1.2 years; BMI, 23.1 ± 2.7 kg/m²) were randomised to one of three test breakfasts, on three separate occasions, separated by 1 week. The breakfasts consisted of eggs on toast, cereal (cornflakes) with milk and toast, or a croissant and orange juice. Subjective ratings of satiety, hunger, fullness and desire to eat were recorded at 30-min intervals by electronic visual analogue scales (VAS). Energy intake was assessed by weighed food intake at an ad libitum lunch and evening meal.Participants showed increased satiety, less hunger and a lower desire to eat after the breakfast containing eggs relative to the cereal (p0.02), and croissant-based meals (p0.0001). The egg breakfast was also accompanied by a significantly lower intake of energy relative to the croissant- and cereal-based breakfasts at the buffet lunch and evening meal, respectively, 1,284 ± 464 (egg) versus 1,442 ± 426 kcal (croissant), p = 0.03, 1,407 ± 379 (cereal) at lunch and 1,899 ± 729 (egg) versus 2,214 ± 620 kcal (cereal), p = 0.02, 2,047 ± 712 (croissant) at evening meal. The breakfast meal with the greatest effect on satiety and subsequent intake of energy was distinct in having the highest protein and lowest carbohydrate content relative to the other two breakfasts.These findings provide evidence to support the importance of food choice at breakfast as a means of increasing satiety in the morning and reducing energy intake at lunch.

Monoclonal antibodies (mAbs) have inaugurated the concepts of tumor-targeted therapy and personalized medicine. A new family of mAbs is currently emerging in the clinic, which target immune cells rather than cancer cells. These immune-targeted therapies have recently demonstrated long-term tumor responses in adults with refractory/relapsing metastatic solid tumors. Pediatric cancers are different from their adult counterparts in terms of histological features and immune infiltrates. However, the same immune checkpoint targets can be expressed within the microenvironment of pediatric tumors. The benefits of immune checkpoint blockade in pediatric cancers are currently under evaluation in early phase clinical trials.

Examines the history of coffee drinking. Describes the constituents of coffee and its physiological effects on the drinker. Reviews the research done in the past on caffeine’s effects on health. Recommends a safe level of consumption at between 300‐400mg of caffeine per day which translates to four or five cups of average strength coffee.