2.1. Study design, patients, and sample inclusion

The iTHER program opened after ethics approval in April of 2017 as a prospective non-interventional observational study (Netherlands Trial Register, Trial NL5728; NL56826.078.16). Fig. 1A depicts the study workflow, which was developed in close international collaboration with INFORM, sharing Standard Operating Procedures and discussing results at the international Molecular Tumour Board. Protocol details and inclusion criteria are summarised in Supplemental Table 1. iTHER was initiated at three sites in the Netherlands: the Máxima in Utrecht, Erasmus Medical Center in Rotterdam, and Amsterdam University Medical Center in Amsterdam. Since June 2018, care for all children with cancer is centralised at the Máxima and therefore iTHER continued as a single-site study. Eligible patients included children, adolescents, and young adults with (suspected) high-risk, relapsed, or refractory malignancies. After written informed consent, fresh tumour biopsy of the current disease episode, or bone marrow in case of leukaemia, as well as germline material was obtained and processed immediately to avoid delay and tissue degradation. Material was evaluated by a dedicated staff pathologist with specific expertise in paediatric oncology and distributed for routine diagnostic testing, and subsequently approved for ongoing research including iTHER, and biobanking. For solid and central nervous system (CNS) tumours included in iTHER, frozen sections were prepared to assess tumour cell percentage by haematoxylin and eosin (H&E) staining, requiring at least 20% tumour content. Clinical data including age, sex, diagnosis, administered therapy, and best response to therapy were entered into the study database.

iTHER program workflow and cohort demographics. (A) Detailed iTHER pipeline is depicted. ∗ After consent, samples are processed and sequenced utilising INFORM and/or Máxima pipeline. Curated and prioritised events are discussed at the Molecular Tumour Board to identify molecularly matched treatment options. ∗ Created with BioRender.com. (B) iTHER cohort consisting of 302 samples from 253 patients with relapsed, refractory, or high-risk newly diagnosed paediatric cancer. The innermost ring visualises disease distribution by the three main categories: extracranial solid tumour (Solid tumour), Central Nervous System tumour (CNS) and Haematopoietic Malignancies (HM). The outer ring represents the frequency of samples within each subtype: neuroblastoma (NBL), osteosarcoma (OS), rhabdomyosarcoma (RMS), other sarcoma (Sarcoma other), other solid tumours (Other ST), Ewing sarcoma, Wilms tumour (Wilms), malignant rhabdoid tumour (MRT) and extracranial germ cell tumour (GCT); high-grade glioma (HGG), other CNS tumours (Other CNS), medulloblastoma (Medullo), Ependymoma; acute lymphoblastic leukaemia (ALL), acute myeloid leukaemia (AML), and lymphoma (Lymphoma). (C) Age distribution of the cohort, highlighting stage of disease: primary high-risk disease, primary refractory disease, relapse, and secondary malignancy.