Development of a targeted sequencing approach to identify prognostic, predictive and diagnostic markers in paediatric solid tumours

// Elisa Izquierdo 1, 2 , Lina Yuan 1 , Sally George 3, 4 , Michael Hubank 1 , Chris Jones 2 , Paula Proszek 1 , Janet Shipley 5 , Susanne A. Gatz 4, 5 , Caedyn Stinson 6 , Andrew S. Moore 6, 7, 8 , Steven C. Clifford 9 , Debbie Hicks 9 , Janet C. Lindsey 9 , Rebecca M. Hill 9 , Thomas S. Jacques 10, 11 , Jane Chalker 12 , Khin Thway 13 , Simon O’Connor 14 , Lynley Marshall 4 , Lucas Moreno 4, 15 , Andrew Pearson 4 , Louis Chesler 3, 4 , Brian A. Walker 1, 16, * and David Gonzalez De Castro 1, 17, * 1 Molecular Diagnostics Department, The Institute of Cancer Research and Clinical Genomics, The Royal Marsden NHS Foundation, London, United Kingdom 2 Glioma Team, Division of Molecular Pathology and Cancer Therapeutics, The Institute of Cancer Research, London, United Kingdom 3 Paediatric Tumour Biology, Division of Molecular Pathology, The Institute of Cancer Research, London, United Kingdom 4 Paediatric Drug Development Team, Children and Young People's Unit, Royal Marsden NHS Foundation Trust, London, United Kingdom 5 Sarcoma Molecular Pathology Team, Divisions of Molecular Pathology and Cancer Therapeutics, The Institute of Cancer Research, London, United Kingdom 6 The University of Queensland Diamantina Institute, Translational Research Institute, Brisbane, Australia 7 Oncology Service, Children’s Health Queensland Hospital and Health Service, Brisbane, Australia 8 UQ Child Health Research Centre, The University of Queensland, Brisbane, Australia 9 Wolfson Childhood Cancer Research Centre, Northern Institute for Cancer Research, Newcastle University, Newcastle Upon Tyne, United Kingdom 10 Department of Histology, Great Ormond Street Hospital for Children NHS Foundation Trust, London, United Kingdom 11 Developmental Biology and Cancer Programme, UCL GOS Institute of Child Health, London, United Kingdom 12 Haematology, Cellular and Molecular Diagnostics Service, UCL GOS Institute of Child Health, London, United Kingdom 13 Sarcoma Unit, Royal Marsden NHS Foundation Trust, London, United Kingdom 14 Haemato-Oncology Unit, Royal Marsden NHS Foundation Trust, London, United Kingdom 15 HNJ-CNIO Clinical Research Unit and Hospital Universitario Niño Jesus, Madrid, Spain 16 Myeloma Institute, University of Arkansas for Medical Sciences, Little Rock, Arkansas, USA 17 Centre for Cancer Research and Cell Biology, Queen's University Belfast, Belfast, United Kingdom * These authors have contributed equally to this work Correspondence to: David Gonzalez De Castro, email: D.GonzalezdeCastro@qub.ac.uk Keywords: childhood cancer; targeted sequencing; molecular diagnostics; panel validation; targeted therapies Received: July 28, 2017      Accepted: November 16, 2017      Published: December 06, 2017 ABSTRACT The implementation of personalised medicine in childhood cancers has been limited by a lack of clinically validated multi-target sequencing approaches specific for paediatric solid tumours. In order to support innovative clinical trials in high-risk patients with unmet need, we have developed a clinically relevant targeted sequencing panel spanning 311 kb and comprising 78 genes involved in childhood cancers. A total of 132 samples were used for the validation of the panel, including Horizon Discovery cell blends (n=4), cell lines (n=15), formalin-fixed paraffin embedded (FFPE, n=83) and fresh frozen tissue (FF, n=30) patient samples. Cell blends containing known single nucleotide variants (SNVs, n=528) and small insertion-deletions (indels n=108) were used to define panel sensitivities of ≥98% for SNVs and ≥83% for indels 95% CI and panel specificity of ≥98% 95% CI for SNVs. FFPE samples performed comparably to FF samples (n=15 paired). Of 95 well-characterised genetic abnormalities in 33 clinical specimens and 13 cell lines (including SNVs, indels, amplifications, rearrangements and chromosome losses), 94 (98.9%) were detected by our approach. We have validated a robust and practical methodology to guide clinical management of children with solid tumours based on their molecular profiles. Our work demonstrates the value of targeted gene sequencing in the development of precision medicine strategies in paediatric oncology.