The Virtual Child.

SUMMARY: We are building the world's first Virtual Child-a computer model of normal and cancerous human development at the level of each individual cell. The Virtual Child will "develop cancer" that we will subject to unlimited virtual clinical trials that pinpoint, predict, and prioritize potential new treatments, bringing forward the day when no child dies of cancer, giving each one the opportunity to lead a full and healthy life.

Targeting pediatric cancers via T-cell recognition of the monomorphic MHC class I-related protein MR1.

Human leukocyte antigen (HLA) restriction of conventional T-cell targeting introduces complexity in generating T-cell therapy strategies for patients with cancer with diverse HLA-backgrounds. A subpopulation of atypical, major histocompatibility complex-I related protein 1 (MR1)-restricted T-cells, distinctive from mucosal-associated invariant T-cells (MAITs), was recently identified recognizing currently unidentified MR1-presented cancer-specific metabolites. It is hypothesized that the MC.7.G5 MR1T-clone has potential as a pan-cancer, pan-population T-cell immunotherapy approach. These cells are irresponsive to healthy tissue while conferring T-cell receptor(TCR) dependent, HLA-independent cytotoxicity to a wide range of adult cancers. Studies so far are limited to adult malignancies. Here, we investigated the potential of MR1-targeting cellular therapy strategies in pediatric cancer. Bulk RNA sequencing data of primary pediatric tumors were analyzed to assess MR1 expression. In vitro pediatric tumor models were subsequently screened to evaluate their susceptibility to engineered MC.7.G5 TCR-expressing T-cells. Targeting capacity was correlated with qPCR-based MR1 mRNA and protein overexpression. RNA expression of MR1 in primary pediatric tumors varied widely within and between tumor entities. Notably, embryonal tumors exhibited significantly lower MR1 expression than other pediatric tumors. In line with this, most screened embryonal tumors displayed resistance to MR1T-targeting in vitro MR1T susceptibility was observed particularly in pediatric leukemia and diffuse midline glioma models. This study demonstrates potential of MC.7.G5 MR1T-cell immunotherapy in pediatric leukemias and diffuse midline glioma, while activity against embryonal tumors was limited. The dismal prognosis associated with relapsed/refractory leukemias and high-grade brain tumors highlights the promise to improve survival rates of children with these cancers.

Tissue Organoid Cultures Metabolize Dietary Carcinogens Proficiently and Are Effective Models for DNA Adduct Formation.

Human tissue three-dimensional (3D) organoid cultures have the potential to reproduce in vitro the physiological properties and cellular architecture of the organs from which they are derived. The ability of organoid cultures derived from human stomach, liver, kidney, and colon to metabolically activate three dietary carcinogens, aflatoxin B1 (AFB1), aristolochic acid I (AAI), and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), was investigated. In each case, the response of a target tissue (liver for AFB1; kidney for AAI; colon for PhIP) was compared with that of a nontarget tissue (gastric). After treatment cell viabilities were measured, DNA damage response (DDR) was determined by Western blotting for p-p53, p21, p-CHK2, and γ-H2AX, and DNA adduct formation was quantified by mass spectrometry. Induction of the key xenobiotic-metabolizing enzymes (XMEs) CYP1A1, CYP1A2, CYP3A4, and NQO1 was assessed by qRT-PCR. We found that organoids from different tissues can activate AAI, AFB1, and PhIP. In some cases, this metabolic potential varied between tissues and between different cultures of the same tissue. Similarly, variations in the levels of expression of XMEs were observed. At comparable levels of cytotoxicity, organoids derived from tissues that are considered targets for these carcinogens had higher levels of adduct formation than a nontarget tissue.

SMARCB1 loss activates patient-specific distal oncogenic enhancers in malignant rhabdoid tumors.

Malignant rhabdoid tumor (MRT) is a highly malignant and often lethal childhood cancer. MRTs are genetically defined by bi-allelic inactivating mutations in SMARCB1, a member of the BRG1/BRM-associated factors (BAF) chromatin remodeling complex. Mutations in BAF complex members are common in human cancer, yet their contribution to tumorigenesis remains in many cases poorly understood. Here, we study derailed regulatory landscapes as a consequence of SMARCB1 loss in the context of MRT. Our multi-omics approach on patient-derived MRT organoids reveals a dramatic reshaping of the regulatory landscape upon SMARCB1 reconstitution. Chromosome conformation capture experiments subsequently reveal patient-specific looping of distal enhancer regions with the promoter of the MYC oncogene. This intertumoral heterogeneity in MYC enhancer utilization is also present in patient MRT tissues as shown by combined single-cell RNA-seq and ATAC-seq. We show that loss of SMARCB1 activates patient-specific epigenetic reprogramming underlying MRT tumorigenesis.

Hallmark discoveries in the biology of Wilms tumour.

The modern study of Wilms tumour was prompted nearly 50 years ago, when Alfred Knudson proposed the 'two-hit' model of tumour development. Since then, the efforts of researchers worldwide have substantially expanded our knowledge of Wilms tumour biology, including major advances in genetics - from cloning the first Wilms tumour gene to high-throughput studies that have revealed the genetic landscape of this tumour. These discoveries improve understanding of the embryonal origin of Wilms tumour, familial occurrences and associated syndromic conditions. Many efforts have been made to find and clinically apply prognostic biomarkers to Wilms tumour, for which outcomes are generally favourable, but treatment of some affected individuals remains challenging. Challenges are also posed by the intratumoural heterogeneity of biomarkers. Furthermore, preclinical models of Wilms tumour, from cell lines to organoid cultures, have evolved. Despite these many achievements, much still remains to be discovered: further molecular understanding of relapse in Wilms tumour and of the multiple origins of bilateral Wilms tumour are two examples of areas under active investigation. International collaboration, especially when large tumour series are required to obtain robust data, will help to answer some of the remaining unresolved questions.

Unbiased transcription factor CRISPR screen identifies ZNF800 as master repressor of enteroendocrine differentiation.

Enteroendocrine cells (EECs) are hormone-producing cells residing in the epithelium of stomach, small intestine (SI), and colon. EECs regulate aspects of metabolic activity, including insulin levels, satiety, gastrointestinal secretion, and motility. The generation of different EEC lineages is not completely understood. In this work, we report a CRISPR knockout screen of the entire repertoire of transcription factors (TFs) in adult human SI organoids to identify dominant TFs controlling EEC differentiation. We discovered ZNF800 as a master repressor for endocrine lineage commitment, which particularly restricts enterochromaffin cell differentiation by directly controlling an endocrine TF network centered on PAX4. Thus, organoid models allow unbiased functional CRISPR screens for genes that program cell fate.

ErbB2 (HER2)-CAR-NK-92 cells for enhanced immunotherapy of metastatic fusion-driven alveolar rhabdomyosarcoma.

Introduction: Metastatic rhabdomyosarcoma (RMS) is a challenging tumor entity that evades conventional treatments and endogenous antitumor immune responses, highlighting the need for novel therapeutic strategies. Applying chimeric antigen receptor (CAR) technology to natural killer (NK) cells may offer safe, effective, and affordable therapies that enhance cancer immune surveillance. Methods: Here, we assess the efficacy of clinically usable CAR-engineered NK cell line NK-92/5.28.z against ErbB2-positive RMS in vitro and in a metastatic xenograft mouse model. Results: Our results show that NK-92/5.28.z cells effectively kill RMS cells in vitro and significantly prolong survival and inhibit tumor progression in mice. The persistence of NK-92/5.28.z cells at tumor sites demonstrates efficient antitumor response, which could help overcome current obstacles in the treatment of solid tumors. Discussion: These findings encourage further development of NK-92/5.28.z cells as off-the-shelf immunotherapy for the treatment of metastatic RMS.

CRISPR-Cas9 Library Screening Identifies Novel Molecular Vulnerabilities in KMT2A-Rearranged Acute Lymphoblastic Leukemia.

In acute lymphoblastic leukemia (ALL), chromosomal translocations involving the KMT2A gene represent highly unfavorable prognostic factors and most commonly occur in patients less than 1 year of age. Rearrangements of the KMT2A gene drive epigenetic changes that lead to aberrant gene expression profiles that strongly favor leukemia development. Apart from this genetic lesion, the mutational landscape of KMT2A-rearranged ALL is remarkably silent, providing limited insights for the development of targeted therapy. Consequently, identifying potential therapeutic targets often relies on differential gene expression, yet the inhibition of these genes has rarely translated into successful therapeutic strategies. Therefore, we performed CRISPR-Cas9 knock-out screens to search for genetic dependencies in KMT2A-rearranged ALL. We utilized small-guide RNA libraries directed against the entire human epigenome and kinome in various KMT2A-rearranged ALL, as well as wild-type KMT2A ALL cell line models. This screening approach led to the discovery of the epigenetic regulators ARID4B and MBD3, as well as the receptor kinase BMPR2 as novel molecular vulnerabilities and attractive therapeutic targets in KMT2A-rearranged ALL.

Systematic discovery of gene fusions in pediatric cancer by integrating RNA-seq and WGS.

BACKGROUND: Gene fusions are important cancer drivers in pediatric cancer and their accurate detection is essential for diagnosis and treatment. Clinical decision-making requires high confidence and precision of detection. Recent developments show RNA sequencing (RNA-seq) is promising for genome-wide detection of fusion products but hindered by many false positives that require extensive manual curation and impede discovery of pathogenic fusions. METHODS: We developed Fusion-sq to overcome existing disadvantages of detecting gene fusions. Fusion-sq integrates and "fuses" evidence from RNA-seq and whole genome sequencing (WGS) using intron-exon gene structure to identify tumor-specific protein coding gene fusions. Fusion-sq was then applied to the data generated from a pediatric pan-cancer cohort of 128 patients by WGS and RNA sequencing. RESULTS: In a pediatric pan-cancer cohort of 128 patients, we identified 155 high confidence tumor-specific gene fusions and their underlying structural variants (SVs). This includes all clinically relevant fusions known to be present in this cohort (30 patients). Fusion-sq distinguishes healthy-occurring from tumor-specific fusions and resolves fusions in amplified regions and copy number unstable genomes. A high gene fusion burden is associated with copy number instability. We identified 27 potentially pathogenic fusions involving oncogenes or tumor-suppressor genes characterized by underlying SVs, in some cases leading to expression changes indicative of activating or disruptive effects. CONCLUSIONS: Our results indicate how clinically relevant and potentially pathogenic gene fusions can be identified and their functional effects investigated by combining WGS and RNA-seq. Integrating RNA fusion predictions with underlying SVs advances fusion detection beyond extensive manual filtering. Taken together, we developed a method for identifying candidate gene fusions that is suitable for precision oncology applications. Our method provides multi-omics evidence for assessing the pathogenicity of tumor-specific gene fusions for future clinical decision making.

Single-cell transcriptomics reveals immune suppression and cell states predictive of patient outcomes in rhabdomyosarcoma.

Paediatric rhabdomyosarcoma (RMS) is a soft tissue malignancy of mesenchymal origin that is thought to arise as a consequence of derailed myogenic differentiation. Despite intensive treatment regimens, the prognosis for high-risk patients remains dismal. The cellular differentiation states underlying RMS and how these relate to patient outcomes remain largely elusive. Here, we use single-cell mRNA sequencing to generate a transcriptomic atlas of RMS. Analysis of the RMS tumour niche reveals evidence of an immunosuppressive microenvironment. We also identify a putative interaction between NECTIN3 and TIGIT, specific to the more aggressive fusion-positive (FP) RMS subtype, as a potential cause of tumour-induced T-cell dysfunction. In malignant RMS cells, we define transcriptional programs reflective of normal myogenic differentiation and show that these cellular differentiation states are predictive of patient outcomes in both FP RMS and the less aggressive fusion-negative subtype. Our study reveals the potential of therapies targeting the immune microenvironment of RMS and suggests that assessing tumour differentiation states may enable a more refined risk stratification.

Oncogenic signaling is coupled to colorectal cancer cell differentiation state.

Colorectal cancer progression is intrinsically linked to stepwise deregulation of the intestinal differentiation trajectory. In this process, sequential mutations of APC, KRAS, TP53, and SMAD4 enable oncogenic signaling and establish the hallmarks of cancer. Here, we use mass cytometry of isogenic human colon organoids and patient-derived cancer organoids to capture oncogenic signaling, cell phenotypes, and differentiation states in a high-dimensional single-cell map. We define a differentiation axis in all tumor progression states from normal to cancer. Our data show that colorectal cancer driver mutations shape the distribution of cells along the differentiation axis. In this regard, subsequent mutations can have stem cell promoting or restricting effects. Individual nodes of the cancer cell signaling network remain coupled to the differentiation state, regardless of the presence of driver mutations. We use single-cell RNA sequencing to link the (phospho-)protein signaling network to transcriptomic states with biological and clinical relevance. Our work highlights how oncogenes gradually shape signaling and transcriptomes during tumor progression.

Atypical teratoid/rhabdoid tumoroids reveal subgroup-specific drug vulnerabilities.

Atypical teratoid/rhabdoid tumors (ATRTs) represent a rare, but aggressive pediatric brain tumor entity. They are genetically defined by alterations in the SWI/SNF chromatin remodeling complex members SMARCB1 or SMARCA4. ATRTs can be further classified in different molecular subgroups based on their epigenetic profiles. Although recent studies suggest that the different subgroups have distinct clinical features, subgroup-specific treatment regimens have not been developed thus far. This is hampered by the lack of pre-clinical in vitro models representative of the different molecular subgroups. Here, we describe the establishment of ATRT tumoroid models from the ATRT-MYC and ATRT-SHH subgroups. We demonstrate that ATRT tumoroids retain subgroup-specific epigenetic and gene expression profiles. High throughput drug screens on our ATRT tumoroids revealed distinct drug sensitivities between and within ATRT-MYC and ATRT-SHH subgroups. Whereas ATRT-MYC universally displayed high sensitivity to multi-targeted tyrosine kinase inhibitors, ATRT-SHH showed a more heterogeneous response with a subset showing high sensitivity to NOTCH inhibitors, which corresponded to high expression of NOTCH receptors. Our ATRT tumoroids represent the first pediatric brain tumor organoid model, providing a representative pre-clinical model which enables the development of subgroup-specific therapies.

Somatic, Genetic and Epigenetic Changes in Nephrogenic Rests and Their Role in the Transformation to Wilms Tumors, a Systematic Review.

OBJECTIVE: To review somatic genetic changes in nephrogenic rests (NR), which are considered to be precursor lesions of Wilms tumors (WT). METHODS: This systematic review is written according to the PRISMA statement. PubMed and EMBASE were systematically searched for articles in the English language studying somatic genetic changes in NR between 1990 and 2022. RESULTS: Twenty-three studies were included in this review, describing 221 NR of which 119 were pairs of NR and WT. Single gene studies showed mutations in WT1 and WTX, but not CTNNB1 to occur in both NR and WT. Studies investigating chromosomal changes showed loss of heterozygosity of 11p13 and 11p15 to occur in both NR and WT, but loss of 7p and 16q occurred in WT only. Methylome-based studies found differential methylation patterns between NR, WT, and normal kidney (NK). CONCLUSIONS: Over a 30-year time frame, few studies have addressed genetic changes in NR, likely hampered by technical and practical limitations. A limited number of genes and chromosomal regions have been implicated in the early pathogenesis of WT, exemplified by their occurrence in NR, including WT1, WTX, and genes located at 11p15. Further studies of NR and corresponding WT are urgently needed.

The tumor microenvironment and immune targeting therapy in pediatric renal tumors.

This review highlights the role of several immunomodulating elements contributing to the tumor microenvironment of various pediatric renal tumors including Wilms tumor. The roles of innate and adaptive immune cells in renal tumors are summarized as well as immunomodulatory cytokines and other proteins. The expression and the predictive role of checkpoint modulators like PD-L1 and immunomodulating proteins like glypican-3, B7-H3, COX-2 are highlighted with a translational view toward potential therapeutic innovations. We further discuss the current state of preclinical models in advancing this field of study. Finally, examples of clinical trials of immunomodulating strategies such as monoclonal antibodies and chimeric antigen receptor T (CAR-T) cells for relapsed/refractory/progressive pediatric renal tumors are described.

Rewiring glucose metabolism improves 5-FU efficacy in p53-deficient/KRASG12D glycolytic colorectal tumors.

Despite the fact that 5-fluorouracil (5-FU) is the backbone for chemotherapy in colorectal cancer (CRC), the response rates in patients is limited to 50%. The mechanisms underlying 5-FU toxicity are debated, limiting the development of strategies to improve its efficacy. How fundamental aspects of cancer, such as driver mutations and phenotypic heterogeneity, relate to the 5-FU response remains obscure. This largely relies on the limited number of studies performed in pre-clinical models able to recapitulate the key features of CRC. Here, we analyzed the 5-FU response in patient-derived organoids that reproduce the different stages of CRC. We find that 5-FU induces pyrimidine imbalance, which leads to DNA damage and cell death in the actively proliferating cancer cells deficient in p53. Importantly, p53-deficiency leads to cell death due to impaired cell cycle arrest. Moreover, we find that targeting the Warburg effect in KRASG12D glycolytic tumor organoids enhances 5-FU toxicity by further altering the nucleotide pool and, importantly, without affecting non-transformed WT cells. Thus, p53 emerges as an important factor in determining the 5-FU response, and targeting cancer metabolism in combination with replication stress-inducing chemotherapies emerges as a promising strategy for CRC treatment.

Molecular Characterization Reveals Subclasses of 1q Gain in Intermediate Risk Wilms Tumors.

Chromosomal alterations have recurrently been identified in Wilms tumors (WTs) and some are associated with poor prognosis. Gain of 1q (1q+) is of special interest given its high prevalence and is currently actively studied for its prognostic value. However, the underlying mutational mechanisms and functional effects remain unknown. In a national unbiased cohort of 30 primary WTs, we integrated somatic SNVs, CNs and SVs with expression data and distinguished four clusters characterized by affected biological processes: muscle differentiation, immune system, kidney development and proliferation. Combined genome-wide CN and SV profiles showed that tumors profoundly differ in both their types of 1q+ and genomic stability and can be grouped into WTs with co-occurring 1p-/1q+, multiple chromosomal gains or CN neutral tumors. We identified 1q+ in eight tumors that differ in mutational mechanisms, subsequent rearrangements and genomic contexts. Moreover, 1q+ tumors were present in all four expression clusters reflecting activation of various biological processes, and individual tumors overexpress different genes on 1q. In conclusion, by integrating CNs, SVs and gene expression, we identified subgroups of 1q+ tumors reflecting differences in the functional effect of 1q gain, indicating that expression data is likely needed for further risk stratification of 1q+ WTs.

Mesenchymal tumor organoid models recapitulate rhabdomyosarcoma subtypes.

Rhabdomyosarcomas (RMS) are mesenchyme-derived tumors and the most common childhood soft tissue sarcomas. Treatment is intense, with a nevertheless poor prognosis for high-risk patients. Discovery of new therapies would benefit from additional preclinical models. Here, we describe the generation of a collection of 19 pediatric RMS tumor organoid (tumoroid) models (success rate of 41%) comprising all major subtypes. For aggressive tumors, tumoroid models can often be established within 4-8 weeks, indicating the feasibility of personalized drug screening. Molecular, genetic, and histological characterization show that the models closely resemble the original tumors, with genetic stability over extended culture periods of up to 6 months. Importantly, drug screening reflects established sensitivities and the models can be modified by CRISPR/Cas9 with TP53 knockout in an embryonal RMS model resulting in replicative stress drug sensitivity. Tumors of mesenchymal origin can therefore be used to generate organoid models, relevant for a variety of preclinical and clinical research questions.

Prevalence of (Epi)genetic Predisposing Factors in a 5-Year Unselected National Wilms Tumor Cohort: A Comprehensive Clinical and Genomic Characterization.

PURPOSE: Wilms tumor (WT) is associated with (epi)genetic predisposing factors affecting a growing number of WT predisposing genes and loci, including those causing Beckwith-Wiedemann spectrum (BWSp) or WT1-related syndromes. To guide genetic counseling and testing, we need insight into the prevalence of WT predisposing (epi)genetic factors. PATIENTS AND METHODS: All children diagnosed with WT in the Netherlands between 2015 and 2020 were referred to a clinical geneticist. Phenotypic data, disease characteristics, and diagnostic test results were collected. If no genetic predisposition was identified by targeted diagnostic testing, germline (trio-)whole-exome sequencing and BWSp testing on normal kidney-derived DNA were offered. RESULTS: A total of 126 cases were analyzed of 128 identified patients. (Epi)genetic predisposing factors were present in 42 of 126 patients (33.3%) on the basis of a molecular diagnosis in blood-derived DNA (n = 26), normal kidney-derived DNA (n = 12), or solely a clinical diagnosis of BWSp (n = 4). Constitutional, heterozygous DIS3L2 variants were identified as a recurrent predisposing factor in five patients (4%), with a second somatic hit in 4 of 5 tumors. Twenty patients (16%) were diagnosed with BWSp while four additional patients without BWSp features harbored chromosome 11p15 methylation defects in normal kidney tissue. Remaining findings included WT1-related syndromes (n = 10), Fanconi anemia (n = 1), neurofibromatosis type 1 (n = 1), and a pathogenic REST variant (n = 1). In addition, (likely) pathogenic variants in adult-onset cancer predisposition genes (BRCA2, PMS2, CHEK2, and MUTYH) were identified in 5 of 56 (8.9%) patients with available whole-exome sequencing data. Several candidate WT predisposition genes were identified, which require further validation. CONCLUSION: (Epi)genetic WT predisposing factors, including mosaic aberrations and recurrent heterozygous DIS3L2 variants, were present in at least 33.3% of patients with WT. On the basis of these results, we encourage standard genetic testing after counseling by a clinical geneticist.

Characteristics and outcome of children with renal tumors in the Netherlands: The first five-year's experience of national centralization.

Around 6% of all childhood malignancies represent renal tumors, of which a majority includes Wilms tumor (WT). Although survival rates have improved over the last decades, specific patients are still at risk for adverse outcome. In the Netherlands, since 2015, pediatric oncology care for renal tumors has been centralized in the Princess Máxima Center for Pediatric Oncology. Here, we describe experiences of the first 5 years of centralized care and explore whether this influences the epidemiological landscape by comparing data with the Netherlands Cancer Registry (NCR). We identified all patients <19 years with a renal mass diagnosed between 01-01-2015 and 31-12-2019 in the Princess Máxima Center. Epidemiology, characteristics and management were analyzed. We identified 164 patients (including 1 patient who refused consent for registration), in our center with a suspicion of a renal tumor. The remaining 163 cases included WT (n = 118)/cystic partially differentiated nephroblastoma (n = 2)/nephrogenic rests only (n = 6) and non-WT (n = 37). In this period, the NCR included 138 children, 1 17-year-old patient was not referred to the Princess Máxima Center. Central radiology review (before starting treatment) was performed in 121/163 patients, and central pathology review in 148/152 patients that underwent surgery. Treatment stratification, according to SIOP/EpSSG protocols was pursued based on multidisciplinary consensus. Preoperative chemotherapy was administered in 133 patients, whereas 19 patients underwent upfront surgery. Surgery was performed in 152 patients, and from 133 biomaterial was stored. Centralization of care for children with renal tumors led to referral of all but 1 new renal tumor cases in the Netherlands, and leads to referral of very rare subtypes not registered in the NCR, that benefit from high quality diagnostics and multidisciplinary decision making. National centralization of care led to enhanced development of molecular diagnostics and other innovation-based treatments for the future.

Normal and tumor-derived organoids as a drug screening platform for tumor-specific drug vulnerabilities.

Patient-derived tumor organoids can be predictive of patient's treatment responses, and normal tissue-derived organoids allow for drug toxicity testing. Combining both types of organoids therefore enables screening for tumor-specific drug vulnerabilities. Here, we provide a detailed protocol for organoid drug screening using, as proof-of-principle, patient-derived malignant rhabdoid tumor organoids. The protocol can be adapted for drug testing on any tumor and/or normal tissue-derived organoid culture. For complete details on the use and execution of this protocol, please refer to Calandrini et al. (2021).