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BACKGROUND: Molecular profiling of the tumour immune microenvironment (TIME) has enabled the rational choice of immunotherapies in some adult cancers. In contrast, the TIME of paediatric cancers is relatively unexplored. We speculated that a more refined appreciation of the TIME in childhood cancers, rather than a reliance on commonly used biomarkers such as tumour mutation burden (TMB), neoantigen load and PD-L1 expression, is an essential prerequisite for improved immunotherapies in childhood solid cancers. METHODS: We combined immunohistochemistry (IHC) with RNA sequencing and whole-genome sequencing across a diverse spectrum of high-risk paediatric cancers to develop an alternative, expression-based signature associated with CD8+ T-cell infiltration of the TIME. Furthermore, we explored transcriptional features of immune archetypes and T-cell receptor sequencing diversity, assessed the relationship between CD8+ and CD4+ abundance by IHC and deconvolution predictions and assessed the common adult biomarkers such as neoantigen load and TMB. RESULTS: A novel 15-gene immune signature, Immune Paediatric Signature Score (IPASS), was identified. Using this signature, we estimate up to 31% of high-risk cancers harbour infiltrating T-cells. In addition, we showed that PD-L1 protein expression is poorly correlated with PD-L1 RNA expression and TMB and neoantigen load are not predictive of T-cell infiltration in paediatrics. Furthermore, deconvolution algorithms are only weakly correlated with IHC measurements of T-cells. CONCLUSIONS: Our data provides new insights into the variable immune-suppressive mechanisms dampening responses in paediatric solid cancers. Effective immune-based interventions in high-risk paediatric cancer will require individualised analysis of the TIME.

Biomarkers which better match anticancer drugs with cancer driver genes hold the promise of improved clinical responses and cure rates. We developed a precision medicine platform of rapid high-throughput drug screening (HTS) and patient-derived xenografting (PDX) of primary tumor tissue, and evaluated its potential for treatment identification among 56 consecutively enrolled high-risk pediatric cancer patients, compared with conventional molecular genomics and transcriptomics. Drug hits were seen in the majority of HTS and PDX screens, which identified therapeutic options for 10 patients for whom no targetable molecular lesions could be found. Screens also provided orthogonal proof of drug efficacy suggested by molecular analyses and negative results for some molecular findings. We identified treatment options across the whole testing platform for 70% of patients. Only molecular therapeutic recommendations were provided to treating oncologists and led to a change in therapy in 53% of patients, of whom 29% had clinical benefit. These data indicate that in vitro and in vivo drug screening of tumor cells could increase therapeutic options and improve clinical outcomes for high-risk pediatric cancer patients.

Brain tumours are the most common solid tumour in children and the leading cause of cancer related death in children. Current treatments include surgery, chemotherapy and radiotherapy. The need for aggressive treatment means many survivors are left with permanent severe disability, physical, intellectual and social. Recent progress in immunotherapy, including genetically engineered T cells with chimeric antigen receptors (CARs) for treating cancer, may provide new avenues to improved outcomes for patients with paediatric brain cancer. In this review we discuss advances in CAR T cell immunotherapy, the major CAR T cell targets that are in clinical and pre-clinical development with a focus on paediatric brain tumours, the paediatric brain tumour microenvironment and strategies used to improve CAR T cell therapy for paediatric tumours.

The Zero Childhood Cancer Program is a precision medicine program to benefit children with poor-outcome, rare, relapsed or refractory cancer. Using tumor and germline whole genome sequencing (WGS) and RNA sequencing (RNAseq) across 252 tumors from high-risk pediatric patients with cancer, we identified 968 reportable molecular aberrations (39.9% in WGS and RNAseq, 35.1% in WGS only and 25.0% in RNAseq only). Of these patients, 93.7% had at least one germline or somatic aberration, 71.4% had therapeutic targets and 5.2% had a change in diagnosis. WGS identified pathogenic cancer-predisposing variants in 16.2% of patients. In 76 central nervous system tumors, methylome analysis confirmed diagnosis in 71.1% of patients and contributed to a change of diagnosis in two patients (2.6%). To date, 43 patients have received a recommended therapy, 38 of whom could be evaluated, with 31% showing objective evidence of clinical benefit. Comprehensive molecular profiling resolved the molecular basis of virtually all high-risk cancers, leading to clinical benefit in some patients.Copyright © 2020, The Author(s), under exclusive licence to Springer Nature America, Inc.

Background and Aims: Advances in our knowledge of the molecular landscape of cancer has led to the development of targeted therapy and personalised anti-cancer treatment. The PRISM trial (ZERO Childhood Cancer Program) evaluates the benefits of a comprehensive precision medicine approach for high-risk paediatric cancer (survival <30%). Method(s): WGS/RNA-seq/methylation results were discussed in a national Molecular Tumour Board (MTB) with 5 tiers of therapeutic recommendations: Tier 1and 2, clinical evidence in same and different cancer type, respectively; Tier 3 and 4, preclinical evidence in same and different cancer type, respectively; Tier 5, consensus opinion. Result(s): 386 patients (43% diagnosis and 57% relapse/refractory) were enrolled over 3 years, with median time to MTB of 6 weeks. A total of 500 recommendations (21% Tier 1, 38% Tier 2, 32% Tier 3, 14% Tier 4 and 5% Tier 5) were made in 261 (67%) patients (median: 1 recommendation/patient; range 1 - 6). 85% were single-agent recommendations and 15% were combination treatments. The PI3K/mTOR, MAPK and cell cycle/cyclin pathway were the 3 most frequently targeted pathways. 87/261 (33%) patients received MTB recommended treatment (median start time 5.3 weeks). Of the first 43 patients who received MTB recommended treatment, 35 could be evaluated for response: 11% complete response (CR), 20% partial response (PR), 40% stable disease (SD) and 29% progressive disease (PD). The duration of SD was >=24 weeks in 9/14 patients. The clinical benefit rate (CR, PR and SD >24 weeks) was 57%. The proportion of tier 1 and 2 recommendations was similar across all response groups. The CR+PR, SD and PD group consisted of 45%, 50% and 50% of tier 1 and 2 recommendations, respectively. Conclusion(s): The ZERO platform provided personalised therapeutic options for 67% of patients and clinical benefit was observed in majority who received the recommended treatment.

INTRODUCTION SJMB12 is a phase 2 clinical trial led by the St. Jude Children’s Research Hospital (St. Jude) that enrolls patients with medulloblastoma based on their biological subgroup. The large cell/anaplastic (LCA) histologic variant has been identified as an important independent risk factor associated with poor outcome. However, the histologic criteria for LCA is subjective, making the distinction between anaplastic and non-anaplastic medulloblastoma difficult in some cases. METHODS Pathological central review was performed at St. Jude. For all patients enrolled in the study to date, concordance was assessed between the initial and central review diagnosis and histologic variant calls made at the Royal Children’s Hospital Melbourne (RCH) and at St. Jude, respectively. RESULTS Since the SJMB12 clinical trial opened locally in 2014, 34 patients were enrolled, and 31 were eligible for this retrospective study. A total of 12 (39%) cases with discordance were identified. The most frequent disagreement was between the designation of LCA (10 cases, 32%). In five cases the tumour was not designated as LCA variant locally. In five cases the initial designation of LCA was refuted centrally. Overall, this led to a change of treatment stratum for four patients (13%). CONCLUSION A high discordance rate exists between neuropathologists in the designation of LCA variant. Differences in interpretation of the subjective histologic criteria and inconsistencies in the material submitted for central review contributed to the discordance. Incorporation of more objective histologic criteria and implementation of unbiased diagnostic tools may improve the generalisability of future risk stratification.

The identification of rearrangements driving expression of neurotrophic receptor tyrosine kinase (NTRK) family kinases in tumors has become critically important because of the availability of effective, specific inhibitor drugs. Whole-genome sequencing (WGS) combined with RNA sequencing (RNA-seq) can identify novel and recurrent expressed fusions. Here we describe three SPECC1L-NTRK fusions identified in two pediatric central nervous system cancers and an extracranial solid tumor using WGS and RNA-seq. These fusions arose either through a simple balanced rearrangement or in the context of a complex chromoplexy event. We cloned the SPECC1L-NTRK2 fusion directly from a patient sample and showed that enforced expression of this fusion is sufficient to promote cytokine-independent survival and proliferation. Cells transformed by SPECC1L-NTRK2 expression are sensitive to a TRK inhibitor drug. We report here that SPECC1L-NTRK fusions can arise in a range of pediatric cancers. Although WGS and RNA-seq are not required to detect NTRK fusions, these techniques may be of benefit when NTRK fusions are not suspected on clinical grounds or not identified by other methods.

BACKGROUND Constitutional Mismatch Repair Deficiency (CMMRD) is a cancer predisposition syndrome caused by biallelic mutations in the mismatch repair pathway, and high-grade glioma (HGG) constitute the most prevalent brain tumours. Pseudoprogression alludes to radiological changes that mimic tumour progression, but are in fact due to other causes such as therapy related inflammation. It can occur as early as three months post treatment. To our knowledge, its characteristics in CMMRD patients has not been reported. METHODS We retrospectively identified seven patients with CMMRD and history of HGG at The Royal Children’s Hospital, Melbourne from 2005 to 2019. Our objective was to review the characteristics of pseudoprogression in this cohort. RESULTS Out of the seven patients, two with constitutional loss of PMS2 demonstrated evidence of pseudoprogression. Patient 1 presented at 16 years old with a cerebellar anaplastic astrocytoma. She developed clinical and radiological progression within two weeks of starting radiotherapy, persisting up to four months after completion. However, six months post radiation she improved without intervention and the tumour remains stable five years post therapy. Patient 2 presented at 17 years old with a midbrain anaplastic astrocytoma, and showed signs of progression four weeks after completion of radiotherapy. She was then treated with Bevacizumab, an anti-VEGFA antibody with remarkable response. She subsequently received Nivolumab, a checkpoint inhibitor with ongoing stable disease for four months. CONCLUSION Our findings showed that pseudoprogression can occur early in the treatment course in CMMRD patients. Identification of this entity is important for appropriate clinical management.

We report two patients with leukaemia driven by the rare CNTRL-FGFR1 fusion oncogene. This fusion arises from a t(8;9)(p12;q33) translocation, and is a rare driver of biphenotypic leukaemia in children. We used RNA sequencing to report novel features of expressed CNTRL-FGFR1, including CNTRL-FGFR1 fusion alternative splicing. From this knowledge, we designed and tested a Droplet Digital PCR assay that detects CNTRL-FGFR1 expression to approximately one cell in 100 000 using fusion breakpoint-specific primers and probes. We also utilised cell-line models to show that effective tyrosine kinase inhibitors, which may be included in treatment regimens for this disease, are only those that block FGFR1 phosphorylation.

Genes encoding TRK are oncogenic drivers in multiple tumour types including infantile fibrosarcoma, papillary thyroid cancer and high-grade gliomas (HGG). TRK fusions have a critical role in tumourigenesis in 40% of infant HGG. Here we report the first case of a TRK fusion-driven HGG treated with larotrectinib-the first selective pan-TRK inhibitor in clinical development. This 3-year-old girl had failed multiple therapies including chemotherapy and radiotherapy. Tumour profiling confirmed an ETV6-NTRK3 fusion. Treatment with larotrectinib led to rapid clinical improvement with near total resolution of primary and metastatic lesions on MRI imaging. This is the first report of a TRK fusion glioma successfully treated with a TRK inhibitor.

We recently reported that atypical teratoid rhabdoid tumors (ATRTs) comprise at least two transcriptional subtypes with different clinical outcomes; however, the mechanisms underlying therapeutic heterogeneity remained unclear. In this study, we analyzed 191 primary ATRTs and 10 ATRT cell lines to define the genomic and epigenomic landscape of ATRTs and identify subgroup-specific therapeutic targets. We found ATRTs segregated into three epigenetic subgroups with distinct genomic profiles, SMARCB1 genotypes, and chromatin landscape that correlated with differential cellular responses to a panel of signaling and epigenetic inhibitors. Significantly, we discovered that differential methylation of a PDGFRB-associated enhancer confers specific sensitivity of group 2 ATRT cells to dasatinib and nilotinib, and suggest that these are promising therapies for this highly lethal ATRT subtype.

Pilocytic astrocytoma (PA) is the most common brain tumor in children but is rare in adults, and hence poorly studied in this age group. We investigated 222 PA and report increased aneuploidy in older patients. Aneuploid genomes were identified in 45% of adult compared with 17% of pediatric PA. Gains were non-random, favoring chromosomes 5, 7, 6 and 11 in order of frequency, and preferentially affecting non-cerebellar PA and tumors with BRAF V600E mutations and not with KIAA1549-BRAF fusions or FGFR1 mutations. Aneuploid PA differentially expressed genes involved in CNS development, the unfolded protein response, and regulators of genomic stability and the cell cycle (MDM2, PLK2),whose correlated programs were overexpressed specifically in aneuploid PA compared to other glial tumors. Thus, convergence of pathways affecting the cell cycle and genomic stability may favor aneuploidy in PA, possibly representing an additional molecular driver in older patients with this brain tumor.