Emerging therapies in Ewing sarcoma.

PURPOSE OF REVIEW: There is an unmet need to improve outcomes for patients for Ewing sarcoma, a rare, aggressive sarcoma with a peak incidence in adolescents and young adults (AYA). Current therapy at diagnosis involves multiagent chemotherapy and local therapy, but despite intensification of treatment, those with metastases at diagnosis and recurrent disease have poor outcomes. RECENT FINDINGS: Improved understanding of Ewing sarcoma biology has identified novel targets with promising activity in Ewing sarcoma patients, including tyrosine kinase inhibitors that are now undergoing evaluation as combination and maintenance therapy. Other emerging therapies include those that target the EWSR1::FLI1 fusion oncoprotein, and act on DNA damage, cell cycle and apoptotic pathways. Immunotherapeutic approaches, particularly CAR-T-cell therapy directed at GD2, also hold promise. Recent collaborative clinical trials that have defined an international standard of care for patients with newly diagnosed Ewing sarcoma and novel platform studies with adaptive designs offer unique opportunities to investigate these therapies inclusive of all ages. SUMMARY: Close international collaboration between clinicians and biologists will allow us to prioritize promising emerging therapies and develop biomarkers to facilitate their incorporation into standard of care and more rapidly translate into benefit for Ewing sarcoma patients.

Protocol for the 'Supporting Young Cancer Survivors who Smoke' study (PRISM): Informing the development of a smoking cessation intervention for childhood, adolescent and young adult cancer survivors in England.

BACKGROUND: Childhood, adolescent and young adult (CAYA) cancer survivors are vulnerable to adverse late-effects. For CAYA cancer survivors, tobacco smoking is the most important preventable cause of ill-health and early death. Yet, effective strategies to support smoking cessation in this group are lacking. The PRISM study aims to undertake multi-method formative research to explore the need for, and if appropriate, inform the future development of an evidence-based and theory-informed tobacco smoking cessation intervention for CAYA cancer survivors. MATERIALS AND METHODS: PRISM involves three phases of: 1) an environmental scan using multiple strategies to identify and examine a) smoking cessation interventions for CAYA cancer survivors that are published in the international literature and b) current smoking cessation services in England that may be available to, or tailorable to, CAYA cancer survivors; 2) a qualitative study involving semi-structured interviews with CAYA cancer survivors (aged 16-29 years and who are current or recent ex-smokers and/or current vapers) to explore their views and experiences of smoking, smoking cessation and vaping; and 3) stakeholder workshops with survivors, healthcare professionals and other stakeholders to consider the potential for a smoking cessation intervention for CAYA cancer survivors and what such an intervention would need to target and change. Findings will be disseminated to patient groups, healthcare professionals and researchers, through conference presentations, journal papers, plain English summaries and social media. DISCUSSION: PRISM will explore current delivery of, perceived need for, and barriers and facilitators to, smoking cessation advice and support to CAYA cancer survivors from the perspective of both survivors and healthcare professionals. A key strength of PRISM is the user involvement throughout the study and the additional exploration of survivors' views on vaping, a behaviour which often co-occurs with smoking. PRISM is the first step in the development of a person-centred, evidence- and theory-based smoking cessation intervention for CAYA cancer survivors who smoke, which if effective, will reduce morbidity and mortality in the CAYA cancer survivor population.

The development and validation of a needs assessment tool for use with YOUng adult survivors of a CentrAl Nervous system tumor (YOU-CAN).

Background: Adolescent and young adult (AYA) survivors of a central nervous system (CNS) tumor represent a vulnerable group who can experience: social isolation, low rates of employment, and achieving independence can be compromised, leading to poorer quality of life compared with survivors of other cancer types. The aim of this study is to develop and evaluate the validity of a needs assessment tool (NAT) for AYA survivors of a CNS tumor. Methods: Items generated using data from 29 qualitative studies and cognitive interviews (n = 8) produced NAT V1.1 (49 items). 128 of 316 eligible participants attending neuro-oncology clinics at 4 NHS sites between June 2022 and March 2023 completed the NAT V1.1 to allow for item reduction and refinement and to evaluate reliability and validity. A pilot study (n = 6) using YOU-CAN in routine follow-up concluded the study. Results: Hierarchical analysis and Rasch analysis identified 18- and 15-items for removal, respectively. YOU-CAN, comprised of the remaining 16 items, demonstrates excellent test-retest reliability (intra-class correlation coefficient, 0.901, n = 40) and sufficient correlation with the European Quality of Life questionnaire and Supportive Care Needs Survey (Pearson r = 0.433 and 0.590, respectively). Pilot testing showed YOU-CAN triggered discussions of unmet needs in consultations and highlighted the importance of multidisciplinary support. Conclusions: YOU-CAN is a valid and reliable instrument containing items related to concerns about physical and emotional health; family and relationships; self-acceptance; and independence. Future efforts should examine YOU-CAN's feasibility, and develop guidance for managing unmet needs. Routine use of YOU-CAN may improve the identification of otherwise undiscussed unmet needs and opportunities to deliver personalized support.

Protocol for the development of a Core Outcome Set (COS) for Adolescents and Young Adults (AYAs) with cancer.

BACKGROUND: Adolescents and young adults (AYAs) with cancer, defined as individuals aged 15-39 years at initial cancer diagnosis, form a unique population; they face age-specific issues as they transition to adulthood. This paper presents the protocol for the development of a core outcome set (COS) for AYAs with cancer. METHODS: The methodological standards from the Core Outcome Measures in Effectiveness Trials (COMET) and the International Consortium for Health Outcomes Measurement (ICHOM) for COS development will guide the development of the COS for AYAs with cancer. The project will consist of the following phases: (1) define the scope of the COS; (2) establish the need for a COS in this field (3) assemble an international, multi-stakeholder working group; (4) develop a detailed protocol; (5) determine "what to measure" (i.e., outcomes); (6) determine "how to measure" (i.e., measures); and (7) determine "case-mix" variables. CONCLUSIONS: The development of a COS for AYAs with cancer will facilitate the implementation of efficient and relevant standards for data collection, both for clinical trials and in routine healthcare, thereby increasing the usefulness of these data to improve the value of the care given to these underserved young cancer patients.

Precision Medicine for Childhood Cancer: Current Limitations and Future Perspectives.

Greater collaboration needed to realize potential of molecular profiling initiatives for pediatric cancers.

Measurement Properties of Patient-Reported Outcome Measures for Adolescent and Young Adult Survivors of a Central Nervous System Tumor: A Systematic Review.

Purpose: To identify and evaluate patient-reported outcome measures (PROMs) for assessing survivorship-related concepts for adolescent and young adult (AYA) survivors of central nervous system (CNS) tumors. Methods: We searched five electronic databases. Two researchers independently screened all titles for inclusion and used consensus-based standards for the selection of health measurement instruments (COSMIN) guidance to grade the quality of evidence for each measurement property. Results: Four studies met eligibility criteria: single-item pain thermometer; single-item fatigue thermometer; 37-item pediatric functional assessment of cancer therapy-brain tumor survivors, measuring quality of life; and 12-item Perceived Barriers Scale to assess barriers to employment. The Perceived Barrier Scale showed high-quality evidence for internal consistency and moderate quality evidence for construct and structural validity. Evidence for the measurement properties of the other PROMs was low-to-moderate quality. Conclusion: We found one PROM with sufficient evidence for good measurement properties to support its use. This warrants development and evaluation of further PROMs to inform ongoing supportive care for this population. Implications for Cancer Survivors: The Perceived Barriers Scale is sufficiently validated and could be considered to guide support for AYA survivors of CNS tumors to achieve their employment goals.

Delivered relative dose intensity in adolescent and young adult germ cell tumours in England: Assessment of data quality and consistency from clinical trials compared to national cancer registration data.

Adolescent and young adults (AYA) with germ cell tumours (GCT) have poorer survival rates than children and many older adults with the same cancers. There are several likely contributing factors to this, including the treatment received. The prognostic benefit of intended dose intensity is well documented in GCT from trials comparing regimens. However, evidence specific to AYA is limited by poor recruitment of AYA to trials and dose delivery outside trials not being well examined. We examined the utility of cancer registration data and a clinical trials dataset to investigate the delivery of relative dose intensity (RDI) in routine National Health Service practice in England, compared to within international clinical trials. Linked data from the Cancer Outcomes and Services Dataset (COSD) and the Systemic Anti-Cancer Therapy (SACT) dataset, and data from four international clinical trials were analysed. Survival over time was described using Kaplan-Meier estimation; overall, by age category, International Germ-Cell Cancer Collaborative Group (IGCCCG) classification, stage, tumour subtype, primary site, ethnicity and deprivation. Cox regression models were used to determine the fully adjusted effect of RDI on mortality risk. The quality of both datasets was critically evaluated and clinically enhanced. RDI was found to be well maintained in all datasets with higher RDIs associated with improved survival outcomes. Real-world data demonstrated several strengths, including population coverage and inclusion of sociodemographic variables and comorbidity. It is limited in GCT however, by the poor completion of data items enabling risk classification of patients and a higher proportion of missing data.

Experiences and Unmet Needs of Adolescent and Young Adult Survivors of a Brain Tumor (Aged 15-39 Years): A Systematic Review and Meta-ethnography.

BACKGROUND: Brain tumors account for 15% of all adolescent and young adult cancers, and survivors are at risk of ongoing late effects that can severely impact their ability to reach independence. Despite follow-up initiatives advocating a personalized approach, survivors continue to experience ongoing sequelae. A better understanding of the survivorship experience is required to ensure services are able to deliver personalized support. OBJECTIVE: The aim of this systematic search and meta-ethnography was to identify and synthesize qualitative studies to better understand the experiences, perspectives, and needs of adolescent and young adult brain tumor survivors. METHODS: Five databases were searched using predefined criteria, studies were independently screened by two researchers, and those meeting inclusion criteria were synthesized. RESULTS: Twenty-seven studies were synthesized, generating 2 main themes, each with subthemes: (1) individual factors impacting resilience, namely, positive coping styles, managing emotions, and family functioning, and (2) cancer-related factors that challenge the individual, namely, living with societal expectations and barriers to coping. CONCLUSION: The conceptual framework illustrates the challenges and resilience of survivors along the continuum from adolescence to adulthood, reflecting the needs of this age group in 1 model, despite it being a time of rapid growth. The lack of awareness of potential physical and emotional late effects challenges individual resilience, which is further challenged when significant milestones cannot be reached. IMPLICATIONS FOR PRACTICE: There is a role for follow-up services to identify and address unmet needs, provide better information to equip survivors to manage late effects, and support families, particularly those who underwent more intensive treatment.

Radiation treatment of benign tumors in NF2-related-schwannomatosis: A national study of 266 irradiated patients showing a significant increase in malignancy/malignant progression.

Background: Radiation treatment of benign tumors in tumor predisposition syndromes is controversial, but short-term studies from treatment centers suggest safety despite apparent radiation-associated malignancy being reported. We determined whether radiation treatment in NF2-related schwannomatosis patients is associated with increased rates of subsequent malignancy (M)/malignant progression (MP). Methods: All UK patients with NF2 were eligible if they had a clinical/molecular diagnosis. Cases were NF2 patients treated with radiation for benign tumors. Controls were matched for treatment location with surgical/medical treatments based on age and year of treatment. Prospective data collection began in 1990 with addition of retrospective cases in 1969. Kaplan-Meier analysis was performed for malignancy incidence and survival. Outcomes were central nervous system (CNS) M/MP (2cm annualized diameter growth) and survival from index tumor treatment. Results: In total, 1345 NF2 patients, 266 (133-Male) underwent radiation treatments between 1969 and 2021 with median first radiotherapy age of 32.9 (IQR = 22.4-46.0). Nine subsequent CNS malignancies/MPs were identified in cases with only 4 in 1079 untreated (P < .001). Lifetime and 20-year CNS M/MP was ~6% in all irradiated patients-(4.9% for vestibular schwannomas [VS] radiotherapy) versus <1% in the non-irradiated population (P < .001/.01). Controls were well matched for age at NF2 diagnosis and treatment (Males = 133%-50%) and had no M/MP in the CNS post-index tumor treatment (P = .0016). Thirty-year survival from index tumor treatment was 45.62% (95% CI = 34.0-56.5) for cases and 66.4% (57.3-74.0) for controls (P = .02), but was nonsignificantly worse for VS radiotherapy. Conclusion: NF2 patients should not be offered radiotherapy as first-line treatment of benign tumors and should be given a frank discussion of the potential 5% excess absolute risk of M/MP.

Comprehensive molecular profiling of sarcomas in adolescent and young adult patients: Results of the EORTC SPECTA-AYA international proof-of-concept study.

BACKGROUND: Adolescent and young adult (AYA) patients with cancer are poorly recruited to molecularly targeted trials and have not witnessed the advances in cancer treatment and survival seen in other age groups. We report here a pan-European proof-of-concept study to identify actionable alterations in some of the worst prognosis AYA cancers: bone and soft tissue sarcomas. DESIGN: Patients aged 12-29 years with newly diagnosed or recurrent, intermediate or high-grade bone and soft tissue sarcomas were recruited from six European countries. Pathological diagnoses were centrally reviewed. Formalin-fixed tissues were analysed by whole exome sequencing, methylation profiling and RNA sequencing and were discussed in a multidisciplinary, international molecular tumour board. RESULTS: Of 71 patients recruited, 48 (median 20 years, range 12-28) met eligibility criteria. Central pathological review confirmed, modified and re-classified the diagnosis in 41, 3, and 4 cases, respectively. Median turnaround time to discussion at molecular tumour board was 8.4 weeks. whole exome sequencing (n = 48), methylation profiling (n = 44, 85%) and RNA sequencing (n = 24, 50%) led to therapeutic recommendations for 81% patients, including 4 with germ line alterations. The most common were for agents targeted towards tyrosine kinases (n = 20 recommendations), DNA repair (n = 18) and the PI3K/mTOR/AKT pathway (n = 15). Recommendations were generally based on weak evidence such as activity in a different tumour type (n = 68, 61%), reflecting the dearth of relevant molecular clinical trial data in the same tumour type. CONCLUSIONS: We demonstrate here that comprehensive molecular profiling of AYA patients' samples is feasible and deliverable in a European programme.

Amplification of the PLAG-family genes-PLAGL1 and PLAGL2-is a key feature of the novel tumor type CNS embryonal tumor with PLAGL amplification.

Pediatric central nervous system (CNS) tumors represent the most common cause of cancer-related death in children aged 0-14 years. They differ from their adult counterparts, showing extensive clinical and molecular heterogeneity as well as a challenging histopathological spectrum that often impairs accurate diagnosis. Here, we use DNA methylation-based CNS tumor classification in combination with copy number, RNA-seq, and ChIP-seq analysis to characterize a newly identified CNS tumor type. In addition, we report histology, patient characteristics, and survival data in this tumor type. We describe a biologically distinct pediatric CNS tumor type (n = 31 cases) that is characterized by focal high-level amplification and resultant overexpression of either PLAGL1 or PLAGL2, and an absence of recurrent genetic alterations characteristic of other pediatric CNS tumor types. Both genes act as transcription factors for a regulatory subset of imprinted genes (IGs), components of the Wnt/ß-Catenin pathway, and the potential drug targets RET and CYP2W1, which are also specifically overexpressed in this tumor type. A derived PLAGL-specific gene expression signature indicates dysregulation of imprinting control and differentiation/development. These tumors occurred throughout the neuroaxis including the cerebral hemispheres, cerebellum, and brainstem, and were predominantly composed of primitive embryonal-like cells lacking robust expression of markers of glial or neuronal differentiation (e.g., GFAP, OLIG2, and synaptophysin). Tumors with PLAGL1 amplification were typically diagnosed during adolescence (median age 10.5 years), whereas those with PLAGL2 amplification were diagnosed during early childhood (median age 2 years). The 10-year overall survival was 66% for PLAGL1-amplified tumors, 25% for PLAGL2-amplified tumors, 18% for male patients, and 82% for female patients. In summary, we describe a new type of biologically distinct CNS tumor characterized by PLAGL1/2 amplification that occurs predominantly in infants and toddlers (PLAGL2) or adolescents (PLAGL1) which we consider best classified as a CNS embryonal tumor and which is associated with intermediate survival. The cell of origin and optimal treatment strategies remain to be defined.

International variation in childhood cancer mortality rates from 2001 to 2015: Comparison of trends in the International Cancer Benchmarking Partnership countries.

Despite improved survival rates, cancer remains one of the most common causes of childhood death. The International Cancer Benchmarking Partnership (ICBP) showed variation in cancer survival for adults. We aimed to assess and compare trends over time in cancer mortality between children, adolescents and young adults (AYAs) and adults in the six countries involved in the ICBP: United Kingdom, Denmark, Australia, Canada, Norway and Sweden. Trends in mortality between 2001 and 2015 in the six original ICBP countries were examined. Age standardised mortality rates (ASR per million) were calculated for all cancers, leukaemia, malignant and benign central nervous system (CNS) tumours, and non-CNS solid tumours. ASRs were reported for children (age 0-14 years), AYAs aged 15 to 39 years and adults aged 40 years and above. Average annual percentage change (AAPC) in mortality rates per country were estimated using Joinpoint regression. For all cancers combined, significant temporal reductions were observed in all countries and all age groups. However, the overall AAPC was greater for children (-2.9; 95% confidence interval = -4.0 to -1.7) compared to AYAs (-1.8; -2.1 to -1.5) and adults aged >40 years (-1.5; -1.6 to -1.4). This pattern was mirrored for leukaemia, CNS tumours and non-CNS solid tumours, with the difference being most pronounced for leukaemia: AAPC for children -4.6 (-6.1 to -3.1) vs AYAs -3.2 (-4.2 to -2.1) and over 40s -1.1 (-1.3 to -0.8). AAPCs varied between countries in children for all cancers except leukaemia, and in adults over 40 for all cancers combined, but not in subgroups. Improvements in cancer mortality rates in ICBP countries have been most marked among children aged 0 to 14 in comparison to 15 to 39 and over 40 year olds. This may reflect better care, including centralised service provision, treatment protocols and higher trial recruitment rates in children compared to older patients.

COVID-19 in children with haematological malignancies.

BACKGROUND: Children with cancer are not at increased risk of severe SARS-CoV-2 infection; however, adults with haematological malignancies have increased risk of severe infections compared with non-haematological malignancies. METHODS: We compared patients with haematological and non-haematological malignancies enrolled in the UK Paediatric Coronavirus Cancer Monitoring Project between 12 March 2020 and 16 February 2021. Children who received stem cell transplantation were excluded. RESULTS: Only 2/62 patients with haematological malignancy had severe/critical infections, with an OR of 0.5 for patients with haematological compared with non-haematological malignancies. INTERPRETATION: Children with haematological malignancies are at no greater risk of severe SARS-CoV-2 infection than those with non-haematological malignancies.

Oral etoposide as a single agent in childhood and young adult cancer in England: Still a poorly evaluated palliative treatment.

BACKGROUND: Oral etoposide is commonly used in palliative treatment of childhood and young adult cancer without robust evidence. We describe a national, unselected cohort of young people in England treated with oral etoposide using routinely collected, population-level data. METHODS: Patients aged under 25 years at cancer diagnosis (1995-2017) with a treatment record of single-agent oral etoposide in the Systemic AntiCancer Dataset (SACT, 2012-2018) were identified, linked to national cancer registry data using NHS number and followed to 5 January 2019. Overall survival (OS) was estimated for all tumours combined and by tumour group. A Cox model was applied accounting for age, sex, tumour type, prior and subsequent chemotherapy. RESULTS: Total 115 patients were identified during the study period. Mean age was 11.8 years at cancer diagnosis and 15.5 years at treatment with oral etoposide. Median OS was 5.5 months from the start of etoposide; 13 patients survived beyond 2 years. Survival was shortest in patients with osteosarcoma (median survival 3.6 months) and longest in CNS embryonal tumours (15.5 months). Across the cohort, a median of one cycle (range one to nine) of etoposide was delivered. OS correlated significantly with tumour type and prior chemotherapy, but not with other variables. CONCLUSIONS: This report is the largest series to date of oral etoposide use in childhood and young adult cancer. Most patients treated in this real world setting died quickly. Despite decades of use, there are still no robust data demonstrating a clear benefit of oral etoposide for survival.

Development of Randomized Trials in Adults with Medulloblastoma-The Example of EORTC 1634-BTG/NOA-23.

Medulloblastoma is a rare brain malignancy. Patients after puberty are rare and bear an intermediate prognosis. Standard treatment consists of maximal resection plus radio-chemotherapy. Treatment toxicity is high and produces disabling long-term side effects. The sonic hedgehog (SHH) subgroup is highly overrepresented in the post-pubertal and adult population and can be targeted by smoothened (SMO) inhibitors. No practice-changing prospective randomized data have been generated in adults. The EORTC 1634-BTG/NOA-23 trial will randomize patients between standard-dose vs. reduced-dosed craniospinal radiotherapy and SHH-subgroup patients between the SMO inhibitor sonidegib (OdomzoTM, Sun Pharmaceuticals Industries, Inc., New York, USA) in addition to standard radio-chemotherapy vs. standard radio-chemotherapy alone to improve outcomes in view of decreased radiotherapy-related toxicity and increased efficacy. We will further investigate tumor tissue, blood, and cerebrospinal fluid as well as magnetic resonance imaging and radiotherapy plans to generate information that helps to further improve treatment outcomes. Given that treatment side effects typically occur late, long-term follow-up will monitor classic side effects of therapy, but also health-related quality of life, cognition, social and professional outcome, and reproduction and fertility. In summary, we will generate unprecedented data that will be translated into treatment changes in post-pubertal patients with medulloblastoma and will help to design future clinical trials.

Adolescents and Young Adults Living With an Uncertain or Poor Cancer Prognosis: The "New" Lost Tribe.

Historically, adolescent and young adult (AYA) patients with cancer, diagnosed for the first time at age 15 through 39 years, have often been identified as a "lost tribe" without a medical "home"; neither pediatric nor adult oncology services were able to provide age-appropriate care to this specific group. Internationally, AYA care programs are being established to bridge the gap between the age-defined healthcare worlds and to address the specific needs of AYAs with cancer. However, AYA care programs mostly focus on improving cure rates and addressing survivorship issues, and direct less attention to the unique needs of those living with an uncertain and/or poor cancer prognosis. Additionally, palliative care services are typically poorly equipped to address the age-specific needs of this group. Given that increasingly more AYAs with an uncertain and/or poor cancer prognosis are gaining life years because of novel treatments, and sometimes even face the prospect of long-term disease control, AYA care programs should address the unique palliative care needs of this "new" lost tribe within AYA oncology. This report provides a definition and description of the AYA population living with an uncertain and/or poor cancer prognosis in terms of epidemiologic, clinical, and psychosocial characteristics and challenges, and provides perspectives for future research and care initiatives. It also highlights the need to comprehensively examine the experience of AYAs who are living with uncertain and/or poor cancer prognosis to adjust best care practices for this unique group.

Disease course of neurofibromatosis type 2: a 30-year follow-up study of 353 patients seen at a single institution.

BACKGROUND: Limited data exist on the disease course of neurofibromatosis type 2 (NF2) to guide clinical trial design. METHODS: A prospective database of patients meeting NF2 diagnostic criteria, reviewed between 1990 and 2020, was evaluated. Follow-up to first vestibular schwannoma (VS) intervention and death was assessed by univariate analysis and stratified by age at onset, era referred, and inheritance type. Interventions for NF2-related tumors were assessed. Cox regression was performed to determine the relationship between individual factors from time of diagnosis to NF2-related death. RESULTS: Three hundred and fifty-three patients were evaluated. During 4643.1 follow-up years from diagnosis to censoring, 60 patients (17.0%) died. The annual mean number of patients undergoing VS surgery or radiotherapy declined, from 4.66 and 1.65, respectively, per 100 NF2 patients in 1990-1999 to 2.11 and 1.01 in 2010-2020, as the number receiving bevacizumab increased (2.51 per 100 NF2 patients in 2010-2020). Five patients stopped bevacizumab to remove growing meningioma or spinal schwannoma. 153/353 (43.3%) had at least one neurosurgical intervention/radiation treatment within 5 years of diagnosis. Patients asymptomatic at diagnosis had longer time to intervention and better survival compared to those presenting with symptoms. Those symptomatically presenting <16 and >40 years had poorer overall survival than those presenting at 26-39 years (P = .03 and P = .02, respectively) but those presenting between 16 and 39 had shorter time to VS intervention. Individuals with de novo constitutional variants had worse survival than those with de novo mosaic or inherited disease (P = .004). CONCLUSION: Understanding disease course improves prognostication, allowing for better-informed decisions about care.

Severity of COVID-19 in children with cancer: Report from the United Kingdom Paediatric Coronavirus Cancer Monitoring Project.

BACKGROUND: Children with cancer are frequently immunocompromised. While children are generally thought to be at less risk of severe SARS-CoV-2 infection than adults, comprehensive population-based evidence for the risk in children with cancer is unavailable. We aimed to produce evidence of the incidence and outcomes from SARS-CoV-2 in children with cancer attending all hospitals treating this population across the UK. METHODS: Retrospective and prospective observational study of all children in the UK under 16 diagnosed with cancer through data collection from all hospitals providing cancer care to this population. Eligible patients tested positive for SARS-CoV-2 on reverse transcription polymerase chain reaction (RT-PCR). The primary end-point was death, discharge or end of active care for COVID-19 for those remaining in hospital. RESULTS: Between 12 March 2020 and 31 July 2020, 54 cases were identified: 15 (28%) were asymptomatic, 34 (63%) had mild infections and 5 (10%) moderate, severe or critical infections. No patients died and only three patients required intensive care support due to COVID-19. Estimated incidence of hospital identified SARS-CoV-2 infection in children with cancer under 16 was 3%. CONCLUSIONS: Children with cancer with SARS-CoV-2 infection do not appear at increased risk of severe infection compared to the general paediatric population. This is reassuring and supports the continued delivery of standard treatment.

Large Extracellular Vesicles Can be Characterised by Multiplex Labelling Using Imaging Flow Cytometry.

Extracellular vesicles (EVs) are heterogeneous in size (30 nm-10 µm), content (lipid, RNA, DNA, protein), and potential function(s). Many isolation techniques routinely discard the large EVs at the early stages of small EV or exosome isolation protocols. We describe here a standardised method to isolate large EVs from medulloblastoma cells and examine EV marker expression and diameter using imaging flow cytometry. Our approach permits the characterisation of each large EVs as an individual event, decorated with multiple fluorescently conjugated markers with the added advantage of visualising each event to ensure robust gating strategies are applied. METHODS: We describe step-wise isolation and characterisation of a subset of large EVs from the medulloblastoma cell line UW228-2 assessed by fluorescent light microscopy, transmission electron microscopy (TEM) and tunable resistance pulse sensing (TRPS). Viability of parent cells was assessed by Annexin V exposure by flow cytometry. Imaging flow cytometry (Imagestream Mark II) identified EVs by direct fluorescent membrane labelling with Cell Mask Orange (CMO) in conjunction with EV markers. A stringent gating algorithm based on side scatter and fluorescence intensity was applied and expression of EV markers CD63, CD9 and LAMP 1 assessed. RESULTS: UW228-2 cells prolifically release EVs of up to 6 µm. We show that the Imagestream Mark II imaging flow cytometer allows robust and reproducible analysis of large EVs, including assessment of diameter. We also demonstrate a correlation between increasing EV size and co-expression of markers screened. CONCLUSIONS: We have developed a labelling and stringent gating strategy which is able to explore EV marker expression (CD63, CD9, and LAMP1) on individual EVs within a widely heterogeneous population. Taken together, data presented here strongly support the value of exploring large EVs in clinical samples for potential biomarkers, useful in diagnostic screening and disease monitoring.