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Ten percent of children with cancer harbour a mutation in a predisposition gene. In children with the kidney cancer, Wilms tumour, the prevalence is as high as 30%. Certain predispositions are associated with defined histological and clinical features, suggesting differences in tumourigenesis. To investigate this, we assembled a cohort of 137 children with Wilms tumour, of whom 71 had a pathogenic germline or mosaic variant. We examined 237 neoplasms (including two secondary leukaemias), utilising WGS, RNA sequencing and genome wide methylation, validating our findings in an independent cohort. Tumour development differed in children harbouring a predisposition, depending on the variant gene and its developmental timing. Differences pervaded the repertoire of driver events, including high risk mutations, the clonal architecture of normal kidneys, and the relatedness of neoplasms from the same individual. Our findings indicate that predisposition may preordain Wilms tumourigenesis, suggesting a variant specific approach to managing children merits consideration.

Therapy-related myeloid neoplasms (t-MN) arise as a complication of chemo- and/or radiotherapy. Although t-MN can occur both in adult and childhood cancer survivors, the mechanisms driving therapy-related leukemogenesis likely vary across different ages. Chemotherapy is thought to induce driver mutations in children, whereas in adults pre-existing mutant clones are selected by the exposure. However, selective pressures induced by chemotherapy early in life are less well studied. Here, we use single-cell whole genome sequencing and phylogenetic inference to show that the founding cell of t-MN in children starts expanding after cessation of platinum exposure. In patients with Li-Fraumeni syndrome, characterized by a germline TP53 mutation, we find that the t-MN already expands during treatment, suggesting that platinum-induced growth inhibition is TP53-dependent. Our results demonstrate that germline aberrations can interact with treatment exposures in inducing t-MN, which is important for the development of more targeted, patient-specific treatment regimens and follow-up., (© 2024. The Author(s).)

Detection of somatic mutations in single cells has been severely hampered by technical limitations of whole-genome amplification. Novel technologies including primary template-directed amplification (PTA) significantly improved the accuracy of single-cell whole-genome sequencing (WGS) but still generate hundreds of artifacts per amplification reaction. We developed a comprehensive bioinformatic workflow, called the PTA Analysis Toolbox (PTATO), to accurately detect single base substitutions, insertions-deletions (indels), and structural variants in PTA-based WGS data. PTATO includes a machine learning approach and filtering based on recurrence to distinguish PTA artifacts from true mutations with high sensitivity (up to 90%), outperforming existing bioinformatic approaches. Using PTATO, we demonstrate that hematopoietic stem cells of patients with Fanconi anemia, which cannot be analyzed using regular WGS, have normal somatic single base substitution burdens but increased numbers of deletions. Our results show that PTATO enables studying somatic mutagenesis in the genomes of single cells with unprecedented sensitivity and accuracy., Competing Interests: The authors declare no competing interests., (© 2023 The Author(s).)

NUP98 fusions comprise a family of rare recurrent alterations in AML, associated with adverse outcomes. In order to define the underlying biology and clinical implications of this family of fusions, we performed comprehensive transcriptome, epigenome, and immunophenotypic profiling of 2,235 children and young adults with AML and identified 160 NUP98 rearrangements (7.2%), including 108 NUP98-NSD1 (4.8%), 32 NUP98-KDM5A (1.4%) and 20 NUP98-X cases (0.9%) with 13 different fusion partners. Fusion partners defined disease characteristics and biology; patients with NUP98-NSD1 or NUP98-KDM5A had distinct immunophenotypic, transcriptomic, and epigenomic profiles. Unlike the two most prevalent NUP98 fusions, NUP98-X variants are typically not cryptic. Furthermore, NUP98-X cases are associated with WT1 mutations, and have epigenomic profiles that resemble either NUP98-NSD1 or NUP98-KDM5A. Cooperating FLT3-ITD and WT1 mutations define NUP98-NSD1, and chromosome 13 aberrations are highly enriched in NUP98-KDM5A. Importantly, we demonstrate that NUP98 fusions portend dismal overall survival, with the noteworthy exception of patients bearing abnormal chromosome 13 (clinicaltrials gov. Identifiers: NCT00002798, NCT00070174, NCT00372593, NCT01371981).

Childhood cancer survivors are confronted with various chronic health conditions like therapy-related malignancies. However, it is unclear how exposure to chemotherapy contributes to the mutation burden and clonal composition of healthy tissues early in life. Here, we studied mutation accumulation in hematopoietic stem and progenitor cells (HSPC) before and after cancer treatment of 24 children. Of these children, 19 developed therapy-related myeloid neoplasms (t-MN). Posttreatment HSPCs had an average mutation burden increase comparable to what treatment-naïve cells accumulate during 16 years of life, with excesses up to 80 years. In most children, these additional mutations were induced by clock-like processes, which are also active during healthy aging. Other patients harbored mutations that could be directly attributed to treatments like platinum-based drugs and thiopurines. Using phylogenetic inference, we demonstrate that most t-MN in children originate after the start of treatment and that leukemic clones become dominant during or directly after chemotherapy exposure., Significance: Our study shows that chemotherapy increases the mutation burden of normal blood cells in cancer survivors. Only few drugs damage the DNA directly, whereas in most patients, chemotherapy-induced mutations are caused by processes similar to those present during normal aging. This article is highlighted in the In This Issue feature, p. 1825., (©2022 The Authors; Published by the American Association for Cancer Research.)

Mutational signatures have been identified in cancer genomes, providing information about the causes of cancer and treatment vulnerabilities. This protocol describes an assay to determine the genotoxic mechanisms underlying these signatures using cord-blood derived hematopoietic stem and progenitor cells (CB-HSPCs). CB-HSPCs have a low mutation background, enabling sensitive detection of mutations. First, CB-HSPCs are exposed in vitro , sorted, and clonally expanded. This expansion enables whole-genome sequencing to detect the mutation load and respective patterns induced during genotoxic exposure. For complete details on the use and execution of this protocol, please refer to de Kanter et al. (2021)., Competing Interests: A.R.H., A.v.L., and R.v.B. are named as inventors on a patent application filed resulting from this work., (© 2022 The Author(s).)

Acquisition of oncogenic mutations with age is believed to be rate limiting for carcinogenesis. However, the incidence of leukemia in children is higher than in young adults. Here we compare somatic mutations across pediatric acute myeloid leukemia (pAML) patient-matched leukemic blasts and hematopoietic stem and progenitor cells (HSPCs), as well as HSPCs from age-matched healthy donors. HSPCs in the leukemic bone marrow have limited genetic relatedness and share few somatic mutations with the cell-of-origin of the malignant blasts, suggesting polyclonal hematopoiesis in pAML patients. Compared to normal HSPCs, a subset of pAML cases harbored more somatic mutations and a distinct composition of mutational process signatures. We hypothesize these cases might have arisen from a more committed progenitor. This subset had better outcomes than pAML cases with mutation burden comparable to age-matched healthy HSPCs. Our study provides insights into the etiology and patient stratification of pAML., Competing Interests: Declaration of interests Authors declare no competing interests.

Children show a higher incidence of leukaemia compared with young adolescents, yet their cells are less damaged because of their young age. Children with Down syndrome (DS) have an even higher risk of developing leukaemia during the first years of life. The presence of a constitutive trisomy of chromosome 21 (T21) in DS acts as a genetic driver for leukaemia development, however, additional oncogenic mutations are required. Therefore, T21 provides the opportunity to better understand leukaemogenesis in children. Here, we describe the increased risk of leukaemia in DS during childhood from a somatic evolutionary view. According to this idea, cancer is caused by a variation in inheritable phenotypes within cell populations that are subjected to selective forces within the tissue context. We propose a model in which the increased risk of leukaemia in DS children derives from higher rates of mutation accumulation, already present during fetal development, which is further enhanced by changes in selection dynamics within the fetal liver niche. This model could possibly be used to understand the rate-limiting steps of leukaemogenesis early in life.