Supplementary Figure 1 shows cell viability assays of various pediatric cancer cell lines.

Supplementary Figure 10 shows the IHC markers tested in MNA and NMNA NB PDXs.

Supplementary Figure 3 shows western blots of DNA damage response markers in two pediatric cancer cell lines.

Supplementary Table 5 lists the quantifications of IHC markers in EWS and OS PDX.

Abstract The molecular tumor board (MTB) is central to precision oncology, providing personalized treatment recommendations based on molecular profiles of patient tumors. Genomics is instrumental for MTBs but often fails to identify clinically actionable targets, a gap that phosphoproteomics can fill. We present the tumor proteome activity status (TOPAS) platform, an end-to-end analysis pipeline that converts terabytes of phosphoproteomic data into patient-specific reports for MTB discussions, focusing on clinically relevant signaling linked to oncogenic mechanisms and therapeutic targets. Designed to scale with growing cohorts, the platform integrates data from 1,998 tumor samples to support patient- and cohort-level hypothesis generation. A web portal handles quality control, calculates TOPAS scores, identifies tumor antigens and immune checkpoints, and offers interactive analyses of differential protein abundance and outlier detection. The TOPAS platform is open source, addresses a critical unmet need and facilitates broader adoption of phosphoproteomics in precision oncology in the future.

Despite comprehensive and multi-modal therapy, outcomes for children and adolescents with rhabdomyosarcoma (RMS) have plateaued over the past four decades. This is not for a lack of progress in the basic and translational studies of RMS. Indeed, advances in animal models and/or patient tissue sample acquisition and analysis have improved our understanding of RMS biology. Large-scale sequencing efforts have generated transcriptomic, genomic, and epigenomic datasets that highlight the heterogeneity of RMS and have the potential to improve prognostication and the application of precision medicine in patients with RMS. However, few of these discoveries have been clinically translated, and limitations to the accessibility, uniformity, and application of these new models and datasets hinder their utility. Here, we discuss how advances in understanding RMS biology, optimization of preclinical models, and strategies for translating basic science discoveries to the clinic can potentially improve outcomes for patients with RMS.

Supplementary Figure 9 shows the IHC markers tested in OS, ARMS and ERMS PDXs.

Supplementary Figure 8 shows the IHC markers tested in EWS PDXs.

Supplementary Table 2 lists characteristics of PDX origin.

Supplementary Figure 13 depicts the correlation of baseline IHC marker expression to treatment response.

Supplementary Figure 5 shows all tumor volume curves of the PDXs used in this study.

Supplementary Table 4 lists the quantifications of IHC markers in RMS PDX.

Some aggressive cancers exhibit a level of rapid genome change and therapy resistance that is difficult to explain. Research over the past decade has shown that extrachromosomal DNA (ecDNA) can be the cause. When oncogenic genetic elements untether from chromosomes and no longer follow Mendelian inheritance, genomic chaos and accelerated evolution ensue, generating unique ecDNA biology and non-traditional therapeutic vulnerabilities distinct from traditional mutation-targeting approaches. Here, we put forward a holistic view where ecDNA is integrated into the broader Hallmarks of Cancer framework to better understand the problem and chart a path forward.

Supplementary Figure 11 displays the quantification of each IHC marker in each PDX tested.

Supplementary Table 3 shows statistics for all tumor volume curves.

Sensitive detection of minimal residual disease (MRD) remains a major unmet need in high-risk neuroblastoma. MYCN amplification, a hallmark of high-risk disease, typically occurs on extrachromosomal DNA (ecDNA), but the potential of ecDNA-associated genomic rearrangements for individualized MRD monitoring has not been fully exploited. Here, we applied neuroblastoma-specific hybrid capture-based panel sequencing to identify patient-unique breakpoints within MYCN amplicons, and used Circle-seq and Nanopore sequencing to resolve the extrachromosomal amplicon structure in representative samples. Analysis of 8 neuroblastoma cell lines and 22 primary tumors identified 69 tumor-specific breakpoints. Those selected for assay development were validated by breakpoint-specific PCR and Sanger sequencing. Breakpoints detected in primary tumors remained detectable at relapse, supporting their stability as MRD markers. Breakpoint-specific real-time quantitative PCR and droplet digital PCR detected these junctions in bone marrow aspirates with high specificity and reached sensitivities down to a tumor DNA fraction of 10^-6. We applied this approach to 53 serial bone marrow aspirates from 14 patients with high-risk neuroblastoma to monitor MRD dynamics, resolving treatment response and molecular persistence. In six samples, breakpoint-positive DNA was detected in bone marrow that was negative by conventional cytology and immunocytology, highlighting the added value of molecular monitoring. Together, these findings establish ecDNA breakpoint-based detection as a strategy for MRD assessment in neuroblastoma, that is, in principle, applicable to any ecDNA-amplified oncogene.

Supplementary Figure 7 depicts the overall survival of each pediatric cancer entity under elimusertib treatment.

Supplementary Figure 12 shows changes in pHH3, yH2AX and Clc3 expression in PDXs of different entities.

Supplementary Table 6 lists the quantifications of IHC markers in NB PDX.

Fibroblast growth factor receptor 1 (FGFR1) is recurrently mutated at p.N546 in neuroblastoma. We examined whether mutant FGFR1 is an oncogenic driver, a predictive biomarker, and an actionable vulnerability in this malignancy. FGFR1 mutations at p.N546 were associated with high-risk disease and rapid tumor progression, resulting in dismal outcome for these patients. Ectopic expression of FGFR1N546K induced constitutive downstream signaling and IL-3-independent growth in Ba/F3 cells, indicating oncogene-addicted proliferation. In FGFR1N546K;MYCNtransgenic mice, neuroblastoma developed within the first days of life, with fatal outcome within 3 weeks, reflecting the devastating clinical phenotypes of patients with FGFR1-mutant, high-risk neuroblastoma. Treatment with FGFR inhibitors impaired proliferation and pathway activation in FGFR1N546K-expressing Ba/F3 and patient-derived FGFR1N546K-mutant neuroblastoma cells and inhibited tumor growth in FGFR1N546K;MYCNtransgenic mice and in a chemotherapy-resistant, patient-derived xenograft mouse model. In addition, partial regression of FGFR1N546K-mutant tumor lesions occurred upon treatment with the FGFR inhibitor futibatinib and low-intensity chemotherapy in a patient with refractory neuroblastoma. Together, our data demonstrate that FGFR1N546K is a strong oncogenic driver in neuroblastoma associated with failure of current standard chemotherapy and suggest potential clinical benefit of FGFR-directed therapies in patients with high-risk mutant FGFR1.