RESUMEN
Forkhead box R2 (FOXR2) is a forkhead transcription factor located on the X chromosome whose expression is normally restricted to the testis. In this study, we performed a pan-cancer analysis of FOXR2 activation across more than 10,000 adult and pediatric cancer samples and found FOXR2 to be aberrantly upregulated in 70% of all cancer types and 8% of all individual tumors. The majority of tumors (78%) aberrantly expressed FOXR2 through a previously undescribed epigenetic mechanism that involves hypomethylation of a novel promoter, which was functionally validated as necessary for FOXR2 expression and proliferation in FOXR2-expressing cancer cells. FOXR2 promoted tumor growth across multiple cancer lineages and co-opted ETS family transcription circuits across cancers. Taken together, this study identifies FOXR2 as a potent and ubiquitous oncogene that is epigenetically activated across the majority of human cancers. The identification of hijacking of ETS transcription circuits by FOXR2 extends the mechanisms known to active ETS transcription factors and highlights how transcription factor families cooperate to enhance tumorigenesis. SIGNIFICANCE: This work identifies a novel promoter that drives aberrant FOXR2 expression and delineates FOXR2 as a pan-cancer oncogene that specifically activates ETS transcriptional circuits across human cancers. See related commentary by Liu and Northcott, p. 2977.
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Factores de Transcripción Forkhead , Neoplasias , Adulto , Carcinogénesis/genética , Proliferación Celular , Niño , Epigénesis Genética , Factores de Transcripción Forkhead/genética , Humanos , Masculino , Neoplasias/genética , Oncogenes/genética , Proteínas Proto-Oncogénicas c-ets/genética , Proteínas Proto-Oncogénicas c-ets/metabolismo , Activación TranscripcionalRESUMEN
Primary lymphomas of the central nervous system (PCNSL) are mainly diffuse large B-cell lymphomas (DLBCLs) confined to the central nervous system (CNS). Molecular drivers of PCNSL have not been fully elucidated. Here, we profile and compare the whole-genome and transcriptome landscape of 51 CNS lymphomas (CNSL) to 39 follicular lymphoma and 36 DLBCL cases outside the CNS. We find recurrent mutations in JAK-STAT, NFkB, and B-cell receptor signaling pathways, including hallmark mutations in MYD88 L265P (67%) and CD79B (63%), and CDKN2A deletions (83%). PCNSLs exhibit significantly more focal deletions of HLA-D (6p21) locus as a potential mechanism of immune evasion. Mutational signatures correlating with DNA replication and mitosis are significantly enriched in PCNSL. TERT gene expression is significantly higher in PCNSL compared to activated B-cell (ABC)-DLBCL. Transcriptome analysis clearly distinguishes PCNSL and systemic DLBCL into distinct molecular subtypes. Epstein-Barr virus (EBV)+ CNSL cases lack recurrent mutational hotspots apart from IG and HLA-DRB loci. We show that PCNSL can be clearly distinguished from DLBCL, having distinct expression profiles, IG expression and translocation patterns, as well as specific combinations of genetic alterations.
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Neoplasias del Sistema Nervioso Central , Infecciones por Virus de Epstein-Barr , Linfoma de Células B Grandes Difuso , Sistema Nervioso Central/metabolismo , Neoplasias del Sistema Nervioso Central/genética , Neoplasias del Sistema Nervioso Central/patología , Genómica , Herpesvirus Humano 4 , Humanos , Linfoma de Células B Grandes Difuso/metabolismoRESUMEN
Long-term complications such as radiation-induced second malignancies occur in a subset of patients following radiation-therapy, particularly relevant in pediatric patients due to the long follow-up period in case of survival. Radiation-induced gliomas (RIGs) have been reported in patients after treatment with cranial irradiation for various primary malignancies such as acute lymphoblastic leukemia (ALL) and medulloblastoma (MB). We perform comprehensive (epi-) genetic and expression profiling of RIGs arising after cranial irradiation for MB (n = 23) and ALL (n = 9). Our study reveals a unifying molecular signature for the majority of RIGs, with recurrent PDGFRA amplification and loss of CDKN2A/B and an absence of somatic hotspot mutations in genes encoding histone 3 variants or IDH1/2, uncovering diagnostic markers and potentially actionable targets.
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Inhibidor p15 de las Quinasas Dependientes de la Ciclina/metabolismo , Inhibidor p16 de la Quinasa Dependiente de Ciclina/metabolismo , Amplificación de Genes , Glioma/genética , Recurrencia Local de Neoplasia/patología , Receptor alfa de Factor de Crecimiento Derivado de Plaquetas/genética , Adolescente , Adulto , Niño , Deleción Cromosómica , Análisis por Conglomerados , Metilación de ADN/genética , Femenino , Regulación Neoplásica de la Expresión Génica , Reordenamiento Génico/genética , Genoma Humano , Glioma/patología , Humanos , Estimación de Kaplan-Meier , Masculino , Persona de Mediana Edad , Radiación , Receptor alfa de Factor de Crecimiento Derivado de Plaquetas/metabolismo , Transcripción Genética , Adulto JovenRESUMEN
INFORM is a prospective, multinational registry gathering clinical and molecular data of relapsed, progressive, or high-risk pediatric patients with cancer. This report describes long-term follow-up of 519 patients in whom molecular alterations were evaluated according to a predefined seven-scale target prioritization algorithm. Mean turnaround time from sample receipt to report was 25.4 days. The highest target priority level was observed in 42 patients (8.1%). Of these, 20 patients received matched targeted treatment with a median progression-free survival of 204 days [95% confidence interval (CI), 99-not applicable], compared with 117 days (95% CI, 106-143; P = 0.011) in all other patients. The respective molecular targets were shown to be predictive for matched treatment response and not prognostic surrogates for improved outcome. Hereditary cancer predisposition syndromes were identified in 7.5% of patients, half of which were newly identified through the study. Integrated molecular analyses resulted in a change or refinement of diagnoses in 8.2% of cases. SIGNIFICANCE: The pediatric precision oncology INFORM registry prospectively tested a target prioritization algorithm in a real-world, multinational setting and identified subgroups of patients benefiting from matched targeted treatment with improved progression-free survival, refinement of diagnosis, and identification of hereditary cancer predisposition syndromes.See related commentary by Eggermont et al., p. 2677.This article is highlighted in the In This Issue feature, p. 2659.
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Neoplasias , Niño , Humanos , Neoplasias/diagnóstico , Neoplasias/tratamiento farmacológico , Neoplasias/genética , Medicina de Precisión , Supervivencia sin Progresión , Estudios Prospectivos , Sistema de RegistrosRESUMEN
BACKGROUND: Pediatric patients with relapsed or refractory disease represent a population with a desperate medical need. The aim of the INFORM (INdividualized Therapy FOr Relapsed Malignancies in Childhood) program is to translate next generation molecular diagnostics into a biomarker driven treatment strategy. The program consists of two major foundations: the INFORM registry providing a molecular screening platform and the INFORM2 series of biomarker driven phase I/II trials. The INFORM2 NivEnt trial aims to determine the recommended phase 2 dose (RP2D) of the combination treatment of nivolumab and entinostat (phase I) and to evaluate activity and safety (phase II). METHODS: This is an exploratory non-randomized, open-label, multinational and multicenter seamless phase I/II trial in children and adolescents with relapsed / refractory or progressive high-risk solid tumors and CNS tumors. The phase I is divided in 2 age cohorts: 12-21 years and 6-11 years and follows a 3 + 3 design with two dose levels for entinostat (2 mg/m2 and 4 mg/m2 once per week) and fixed dose nivolumab (3 mg/kg every 2 weeks). Patients entering the trial on RP2D can seamlessly enter phase II which consists of a biomarker defined four group basket trial: high mutational load (group A), high PD-L1 mRNA expression (group B), focal MYC(N) amplification (group C), low mutational load and low PD-L1 mRNA expression and no MYC(N) amplification (group D). A Bayesian adaptive design will be used to early stop cohorts that fail to show evidence of activity. The maximum number of patients is 128. DISCUSSION: This trial intends to exploit the immune enhancing effects of entinostat on nivolumab using an innovative biomarker driven approach in order to maximize the chance of detecting signs of activity. It prevents exposure to unnecessary risks by applying the Bayesian adaptive design for early stopping for futility. The adaptive biomarker driven design provides an innovative approach accelerating drug development and reducing exposure to investigational treatments in these vulnerable children at the same time. TRIAL REGISTRATION: ClinicalTrials.gov, NCT03838042. Registered on 12 February 2019.
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Protocolos de Quimioterapia Combinada Antineoplásica/administración & dosificación , Benzamidas/administración & dosificación , Biomarcadores de Tumor/análisis , Neoplasias/tratamiento farmacológico , Nivolumab/administración & dosificación , Piridinas/administración & dosificación , Adolescente , Protocolos de Quimioterapia Combinada Antineoplásica/efectos adversos , Teorema de Bayes , Benzamidas/efectos adversos , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Niño , Relación Dosis-Respuesta a Droga , Monitoreo de Drogas/métodos , Resistencia a Antineoplásicos , Femenino , Humanos , Masculino , Inutilidad Médica , Mutación , Neoplasias/diagnóstico , Neoplasias/genética , Neoplasias/patología , Nivolumab/efectos adversos , Medicina de Precisión/métodos , Piridinas/efectos adversos , Resultado del Tratamiento , Adulto JovenAsunto(s)
Terapia Genética/efectos adversos , Genómica , Neoplasias Primarias Secundarias/etiología , Leucemia-Linfoma Linfoblástico de Células T Precursoras/etiología , Biomarcadores de Tumor , Perfilación de la Expresión Génica , Pruebas Genéticas , Terapia Genética/métodos , Genómica/métodos , Humanos , Inmunofenotipificación , Neoplasias Primarias Secundarias/diagnóstico , Polimorfismo de Nucleótido Simple , Leucemia-Linfoma Linfoblástico de Células T Precursoras/diagnóstico , Secuenciación del Exoma , Síndrome de Wiskott-Aldrich/complicaciones , Síndrome de Wiskott-Aldrich/genética , Síndrome de Wiskott-Aldrich/terapiaRESUMEN
PURPOSE: Diffuse intrinsic pontine glioma (DIPG) is a highly aggressive paediatric brain tumour with fatal outcome. The Individualised Therapy For Relapsed Malignancies In Childhood (INFORM) registry study offers comprehensive molecular profiling of high-risk tumours to identify target alterations for potential precision therapy. We analysed molecular characteristics and clinical data after brainstem biopsy of all enrolled newly diagnosed DIPGs. PATIENTS AND METHODS: From -February 2015 to February 2018, 21 subsequent primary DIPG cases were enrolled in the nation-wide multicentre INFORM registry study after brainstem biopsy. Whole-genome, whole-exome sequencing and DNA methylation analysis were performed, and RNA-sequencing was added in case of sufficient material. Clinical data were obtained from standardised questionnaires and the INFORM clinical data bank. RESULTS: Tumour material obtained from brainstem biopsy was sufficient for DNA analysis in all cases and RNA analysis in 16 of 21 cases. In 16 of 21 cases (76%), potential targetable alterations were identified including highly relevant MET and NTRK1 fusions as well as an EZH2 alteration not previously described in DIPG. In 5 of 21 cases, molecular information was used for initiation of targeted treatment. The majority of patients (19/21) presented with neurological deficits at diagnosis. Newly arising or worsening of neurological deficits post-biopsy occurred in nine patients. Symptoms were reversible or improved notably in eight cases. CONCLUSION: In this multicentre study setting, brainstem biopsy of DIPG was feasible and yielded sufficient material for comprehensive molecular profiling. Relevant molecular targets were identified impacting clinical management in a substantial subset. Death or severe bleeding occurred in none of the cases. One of 20 patients experienced unilateral paraesthesia possibly related to biopsy.
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Biopsia/métodos , Neoplasias del Tronco Encefálico/cirugía , Glioma/cirugía , Adolescente , Niño , Preescolar , Femenino , Humanos , Masculino , Medicina de Precisión , Estudios ProspectivosRESUMEN
Incomplete understanding of the metastatic process hinders personalized therapy. Here we report the most comprehensive whole-genome study of colorectal metastases vs. matched primary tumors. 65% of somatic mutations originate from a common progenitor, with 15% being tumor- and 19% metastasis-specific, implicating a higher mutation rate in metastases. Tumor- and metastasis-specific mutations harbor elevated levels of BRCAness. We confirm multistage progression with new components ARHGEF7/ARHGEF33. Recurrently mutated non-coding elements include ncRNAs RP11-594N15.3, AC010091, SNHG14, 3' UTRs of FOXP2, DACH2, TRPM3, XKR4, ANO5, CBL, CBLB, the latter four potentially dual protagonists in metastasis and efferocytosis-/PD-L1 mediated immunosuppression. Actionable metastasis-specific lesions include FAT1, FGF1, BRCA2, KDR, and AKT2-, AKT3-, and PDGFRA-3' UTRs. Metastasis specific mutations are enriched in PI3K-Akt signaling, cell adhesion, ECM and hepatic stellate activation genes, suggesting genetic programs for site-specific colonization. Our results put forward hypotheses on tumor and metastasis evolution, and evidence for metastasis-specific events relevant for personalized therapy.
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Adenocarcinoma/genética , Neoplasias Colorrectales/genética , Neoplasias Hepáticas/genética , Medicina de Precisión , Regiones no Traducidas 3'/genética , Proteínas Adaptadoras Transductoras de Señales/genética , Adenocarcinoma/secundario , Anciano , Anoctaminas/genética , Proteínas Reguladoras de la Apoptosis , Proteína BRCA2/genética , Adhesión Celular/genética , Neoplasias Colorrectales/patología , Neoplasias Colorrectales/terapia , Proteínas de Unión al ADN , Matriz Extracelular/genética , Femenino , Factores de Transcripción Forkhead/genética , Células Estrelladas Hepáticas/metabolismo , Humanos , Neoplasias Hepáticas/secundario , Masculino , Proteínas de la Membrana , Proteínas de Transporte de Membrana/genética , Persona de Mediana Edad , Metástasis de la Neoplasia , Proteínas Nucleares/genética , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-cbl/genética , ARN no Traducido , Receptor alfa de Factor de Crecimiento Derivado de Plaquetas/genética , Factores de Intercambio de Guanina Nucleótido Rho/genética , Transducción de Señal , Canales Catiónicos TRPM/genética , Factores de Transcripción/genética , Receptor 2 de Factores de Crecimiento Endotelial Vascular/genética , Secuenciación Completa del GenomaRESUMEN
Brain tumors are the leading cause of cancer-related death in children. Genomic studies have provided insights into molecular subgroups and oncogenic drivers of pediatric brain tumors that may lead to novel therapeutic strategies. To evaluate new treatments, better preclinical models adequately reflecting the biological heterogeneity are needed. Through the Children's Oncology Group ACNS02B3 study, we have generated and comprehensively characterized 30 patient-derived orthotopic xenograft models and seven cell lines representing 14 molecular subgroups of pediatric brain tumors. Patient-derived orthotopic xenograft models were found to be representative of the human tumors they were derived from in terms of histology, immunohistochemistry, gene expression, DNA methylation, copy number, and mutational profiles. In vivo drug sensitivity of targeted therapeutics was associated with distinct molecular tumor subgroups and specific genetic alterations. These models and their molecular characterization provide an unprecedented resource for the cancer community to study key oncogenic drivers and to evaluate novel treatment strategies.
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Bancos de Muestras Biológicas , Neoplasias Encefálicas/patología , Inmunohistoquímica , Ensayos Antitumor por Modelo de Xenoinjerto/métodos , Animales , Línea Celular Tumoral , Niño , Preescolar , Metilación de ADN/genética , Femenino , Genómica , Humanos , Masculino , Ratones , Mutación , PediatríaRESUMEN
Thrombospondins (TSPs) are multidomain glycoproteins with complex matricellular functions in tissue homeostasis and remodeling. We describe a novel role of TSP as a Wnt signaling target in the basal eumetazoan Hydra. Proteome analysis identified Hydra magnipapillata TSP (HmTSP) as a major component of the cnidarian mesoglea. In general, the domain organization of cnidarian TSPs is related to the pentameric TSPs of bilaterians, and in phylogenetic analyses cnidarian TSPs formed a separate clade of high sequence diversity. HmTSP expression in polyps was restricted to the hypostomal tip and tentacle bases that harbor Wnt-regulated organizer tissues. In the hypostome, HmTSP- and Wnt3-expressing cells were identical or in close vicinity to each other, and regions of ectopic tentacle formation induced by pharmacological ß-Catenin activation (Alsterpaullone) corresponded to foci of HmTSP expression. Chromatin immunoprecipitation (ChIP) confirmed binding of Hydra TCF to conserved elements in the HmTSP promotor region. Accordingly, ß-Catenin knockdown by siRNAs reduced normal HmTSP expression at the head organizer. In contrast, knockdown of HmTSP expression led to increased numbers of ectopic organizers in Alsterpaullone-treated animals, indicating a negative regulatory function. Our data suggest an unexpected role for HmTSP as a feedback inhibitor of Wnt signaling during Hydra body axis patterning and maintenance.
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Hydra/metabolismo , Proteoma/metabolismo , Trombospondinas/metabolismo , Animales , Tipificación del Cuerpo/genética , Tipificación del Cuerpo/fisiología , Regiones Promotoras Genéticas/genética , Proteoma/genética , ARN Interferente Pequeño/genética , Transducción de Señal/genética , Transducción de Señal/fisiología , Trombospondinas/genética , Proteínas Wnt/genética , Proteínas Wnt/metabolismo , beta Catenina/genética , beta Catenina/metabolismoRESUMEN
In this Article, author Benedikt Brors was erroneously associated with affiliation number '8' (Department of Developmental Neurobiology, St Jude Children's Research Hospital, Memphis, Tennessee, USA); the author's two other affiliations (affiliations '3' and '7', both at the German Cancer Research Center (DKFZ)) were correct. This has been corrected online.
RESUMEN
Pan-cancer analyses that examine commonalities and differences among various cancer types have emerged as a powerful way to obtain novel insights into cancer biology. Here we present a comprehensive analysis of genetic alterations in a pan-cancer cohort including 961 tumours from children, adolescents, and young adults, comprising 24 distinct molecular types of cancer. Using a standardized workflow, we identified marked differences in terms of mutation frequency and significantly mutated genes in comparison to previously analysed adult cancers. Genetic alterations in 149 putative cancer driver genes separate the tumours into two classes: small mutation and structural/copy-number variant (correlating with germline variants). Structural variants, hyperdiploidy, and chromothripsis are linked to TP53 mutation status and mutational signatures. Our data suggest that 7-8% of the children in this cohort carry an unambiguous predisposing germline variant and that nearly 50% of paediatric neoplasms harbour a potentially druggable event, which is highly relevant for the design of future clinical trials.
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Genoma Humano/genética , Genómica , Mutación/genética , Neoplasias/clasificación , Neoplasias/genética , Adolescente , Adulto , Niño , Cromotripsis , Estudios de Cohortes , Variaciones en el Número de Copia de ADN/genética , Diploidia , Predisposición Genética a la Enfermedad/genética , Mutación de Línea Germinal/genética , Humanos , Terapia Molecular Dirigida , Tasa de Mutación , Neoplasias/tratamiento farmacológico , Proteína p53 Supresora de Tumor/genética , Adulto JovenRESUMEN
Background: O6-methylguanine-DNA methyltransferase (MGMT) promoter methylation status is a predictive biomarker in glioblastoma patients. Glioblastoma without hypermethylated MGMT promoter is largely resistant to treatment with temozolomide. These patients are in particular need of new treatment approaches, which are offered by biomarker-driven clinical trials with targeted drugs based on molecular characterization of individual tumors. Methods: In preparation for an upcoming clinical study, a comprehensive molecular profiling approach was undertaken on tissues from 43 glioblastoma patients harboring an unmethylated MGMT promoter at diagnosis. The diagnostic pipeline covered various levels of molecular characteristics, including whole-exome sequencing, low-coverage whole-genome sequencing, RNA sequencing, as well as microarray-based gene expression profiling and DNA methylation arrays. Results: Complex multilayer molecular diagnostics were feasible in this setting with a median turnaround time of 4-5 weeks from surgery to the molecular tumor board. In 35% of cases, potentially relevant therapeutic decisions were derived from the data. Alterations were most frequently found in receptor tyrosine kinases, members of the phosphoinositide 3-kinase/Akt/mechanistic target of rapamycin and mitogen-activated protein kinase pathway as well as cell cycle control and p53 regulation cascades. Individual tumors harbored clonal alterations such as oncogenic fusions of tyrosine kinases which constitute promising targets for targeted therapies. A prioritization algorithm is proposed to allocate patients with multiple targets to the potentially best treatment option. Conclusion: With this feasibility study, a comprehensive molecular profiling approach for patients with newly diagnosed glioblastoma harboring an unmethylated MGMT promoter is presented. Analyses in this pilot cohort serve as a basis for trials based on targetable alterations and on the question of allocation of patients to the best treatment arm.
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Biomarcadores de Tumor/genética , Neoplasias Encefálicas/genética , Metilación de ADN , Metilasas de Modificación del ADN/genética , Enzimas Reparadoras del ADN/genética , Regulación Neoplásica de la Expresión Génica , Glioblastoma/genética , Regiones Promotoras Genéticas , Proteínas Supresoras de Tumor/genética , Algoritmos , Neoplasias Encefálicas/diagnóstico , Estudios de Factibilidad , Femenino , Estudios de Seguimiento , Glioblastoma/diagnóstico , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Masculino , Persona de Mediana Edad , Proyectos Piloto , Polimorfismo de Nucleótido Simple , Pronóstico , Secuenciación del Exoma , Secuenciación Completa del GenomaRESUMEN
A hierarchically organized cell compartment drives colorectal cancer (CRC) progression. Genetic barcoding allows monitoring of the clonal output of tumorigenic cells without prospective isolation. In this study, we asked whether tumor clone-initiating cells (TcICs) were genetically heterogeneous and whether differences in self-renewal and activation reflected differential kinetics among individual subclones or functional hierarchies within subclones. Monitoring genomic subclone kinetics in three patient tumors and corresponding serial xenografts and spheroids by high-coverage whole-genome sequencing, clustering of genetic aberrations, subclone combinatorics, and mutational signature analysis revealed at least two to four genetic subclones per sample. Long-term growth in serial xenografts and spheroids was driven by multiple genomic subclones with profoundly differing growth dynamics and hence different quantitative contributions over time. Strikingly, genetic barcoding demonstrated stable functional heterogeneity of CRC TcICs during serial xenografting despite near-complete changes in genomic subclone contribution. This demonstrates that functional heterogeneity is, at least frequently, present within genomic subclones and independent of mutational subclone differences.
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Neoplasias Colorrectales/genética , Variaciones en el Número de Copia de ADN , Células Madre Neoplásicas/metabolismo , Esferoides Celulares/metabolismo , Animales , Células Clonales/metabolismo , Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/patología , Análisis Mutacional de ADN/métodos , Heterogeneidad Genética , Genómica/métodos , Humanos , Subunidad gamma Común de Receptores de Interleucina/deficiencia , Subunidad gamma Común de Receptores de Interleucina/genética , Ratones Endogámicos NOD , Ratones Noqueados , Ratones SCID , Mutación , Trasplante Heterólogo , Células Tumorales CultivadasRESUMEN
The 'Individualized Therapy for Relapsed Malignancies in Childhood' (INFORM) precision medicine study is a nationwide German program for children with high-risk relapsed/refractory malignancies, which aims to identify therapeutic targets on an individualised basis. In a pilot phase, reported here, we developed the logistical and analytical pipelines necessary for rapid and comprehensive molecular profiling in a clinical setting. Fifty-seven patients from 20 centers were prospectively recruited. Malignancies investigated included sarcomas (n = 25), brain tumours (n = 23), and others (n = 9). Whole-exome, low-coverage whole-genome, and RNA sequencing were complemented with methylation and expression microarray analyses. Alterations were assessed for potential targetability according to a customised prioritisation algorithm and subsequently discussed in an interdisciplinary molecular tumour board. Next-generation sequencing data were generated for 52 patients, with the full analysis possible in 46 of 52. Turnaround time from sample receipt until first report averaged 28 d. Twenty-six patients (50%) harbored a potentially druggable alteration with a prioritisation score of 'intermediate' or higher (level 4 of 7). Common targets included receptor tyrosine kinases, phosphoinositide 3-kinase-mammalian target of rapamycin pathway, mitogen-activated protein kinase pathway, and cell cycle control. Ten patients received a targeted therapy based on these findings, with responses observed in some previously treatment-refractory tumours. Comparative primary relapse analysis revealed substantial tumour evolution as well as one case of unsuspected secondary malignancy, highlighting the importance of re-biopsy at relapse. This study demonstrates the feasibility of comprehensive, real-time molecular profiling for high-risk paediatric cancer patients. This extended proof-of-concept, with examples of treatment consequences, expands upon previous personalised oncology endeavors, and presents a model with considerable interest and practical relevance in the burgeoning era of personalised medicine.