Repurposing Vandetanib plus Everolimus for the Treatment of ACVR1-Mutant Diffuse Intrinsic Pontine Glioma.

Somatic mutations in ACVR1 are found in a quarter of children with diffuse intrinsic pontine glioma (DIPG), but there are no ACVR1 inhibitors licensed for the disease. Using an artificial intelligence-based platform to search for approved compounds for ACVR1-mutant DIPG, the combination of vandetanib and everolimus was identified as a possible therapeutic approach. Vandetanib, an inhibitor of VEGFR/RET/EGFR, was found to target ACVR1 (K d = 150 nmol/L) and reduce DIPG cell viability in vitro but has limited ability to cross the blood-brain barrier. In addition to mTOR, everolimus inhibited ABCG2 (BCRP) and ABCB1 (P-gp) transporters and was synergistic in DIPG cells when combined with vandetanib in vitro. This combination was well tolerated in vivo and significantly extended survival and reduced tumor burden in an orthotopic ACVR1-mutant patient-derived DIPG xenograft model. Four patients with ACVR1-mutant DIPG were treated with vandetanib plus an mTOR inhibitor, informing the dosing and toxicity profile of this combination for future clinical studies. SIGNIFICANCE: Twenty-five percent of patients with the incurable brainstem tumor DIPG harbor somatic activating mutations in ACVR1, but there are no approved drugs targeting the receptor. Using artificial intelligence, we identify and validate, both experimentally and clinically, the novel combination of vandetanib and everolimus in these children based on both signaling and pharmacokinetic synergies.This article is highlighted in the In This Issue feature, p. 275

Molecular profiling of cell free DNA in patients with paediatric solid tumours

Tumour tissue profiling studies show that a substantial proportion of paediatric cancer patients have potentially actionable alterations and minimally invasive molecular profiling test using cell free DNA (cfDNA) could provide a powerful platform to guide clinical decision-making and to deliver precision treatments. Clinical diagnostic sequencing of cfDNA is well advanced for adult patients, but application to paediatric cancer patients lags behind. To fill this gap, in this thesis I have described the development and validation of a clinically relevant pan-paediatric solid tumour NGS capture panel optimised for cfDNA analysis and an accompanying workflow with low coverage WGS (lcWGS) to molecularly profile paediatric patients with solid tumours. I applied this wrokflow to cfDNA samples from multiple clinical trials and showed that it is informative and yields comparable results to tissue biopsy molecular profiling in patients with extracranial tumours. Additionally, high numbers of cfDNA unique variants detected in patients at relapse showed the potential to complement tissue biopsy testing in many clinical diagnostics situations by allowing detection of tumour heterogeneity and identifying variants missed by tissue biopsy profiling, some of which are potentially targetable or aiding enrolment to clinical trials. In patients with CNS tumours plasma based cfDNA profiling was of limited success, however I showed potential to use cerebrospinal fluid cfDNA instead. Significant changes were observed between diagnostic and primary tissue biopsies with accumulation of SNVs and copy number changes at relapse and the ability to detect these changes in cfDNA was shown in patients with good purity ctDNA, highlighting the potential of cfDNA profiling to monitor tumour evolution in a minimally invasive way. Finally, several case studies showed the potential to track disease progress and identify relapse earlier than conventional methods using longitudinal cfDNA sampling. Overall, in this thesis I have shown that cfDNA is a good biomarker in paediatric cancer care and future molecularly enriched biomarker-driven interventional clinical trials utilising cfDNA are warranted

Liquid biopsy for children with central nervous system tumours: Clinical integration and technical considerations.

Circulating cell-free DNA (cfDNA) analysis has the potential to revolutionise the care of patients with cancer and is already moving towards standard of care in some adult malignancies. Evidence for the utility of cfDNA analysis in paediatric cancer patients is also accumulating. In this review we discuss the limitations of blood-based assays in patients with brain tumours and describe the evidence supporting cerebrospinal fluid (CSF) cfDNA analysis. We make recommendations for CSF cfDNA processing to aid the standardisation and technical validation of future assays. We discuss the considerations for interpretation of cfDNA analysis and highlight promising future directions. Overall, cfDNA profiling shows great potential as an adjunct to the analysis of biopsy tissue in paediatric cancer patients, with the potential to provide a genetic molecular profile of the tumour when tissue biopsy is not feasible. However, to fully realise the potential of cfDNA analysis for children with brain tumours larger prospective studies incorporating serial CSF sampling are required

Circulating tumour DNA sequencing to determine therapeutic response and identify tumour heterogeneity in patients with paediatric solid tumours.

ObjectiveClinical diagnostic sequencing of circulating tumour DNA (ctDNA) is well advanced for adult patients, but application to paediatric cancer patients lags behind.MethodsTo address this, we have developed a clinically relevant (67 gene) NGS capture panel and accompanying workflow that enables sensitive and reliable detection of low-frequency genetic variants in cell-free DNA (cfDNA) from children with solid tumours. We combined gene panel sequencing with low pass whole-genome sequencing of the same library to inform on genome-wide copy number changes in the blood.ResultsAnalytical validity was evaluated using control materials, and the method was found to be highly sensitive (0.96 for SNVs and 0.97 for INDEL), specific (0.82 for SNVs and 0.978 for INDEL), repeatable (>0.93 [95% CI: 0.89-0.95]) and reproducible (>0.87 [95% CI: 0.87-0.95]). Potential for clinical application was demonstrated in 39 childhood cancer patients with a spectrum of solid tumours in which the single nucleotide variants expected from tumour sequencing were detected in cfDNA in 94.4% (17/18) of cases with active extracranial disease. In 13 patients, where serial samples were available, we show a close correlation between events detected in cfDNA and treatment response, demonstrate that cfDNA analysis could be a useful tool to monitor disease progression, and show cfDNA sequencing has the potential to identify targetable variants that were not detected in tumour samples.ConclusionsThis is the first pan-cancer DNA sequencing panel that we know to be optimised for cfDNA in children for blood-based molecular diagnostics in paediatric solid tumours