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In vitro models represent a critical tool in cancer research to study tumor biology and to evaluate new treatment options. Unfortunately, there are no effective preclinical models available that represent Wilms tumor (WT) — the most common pediatric renal tumor. Especially the high-risk blastemal WT subtype is not represented by the few primary cell lines established until now. Here, we describe a new 3D approach for in vitro cultivation of blastemal WT cells, where primary cultures grown in suspension as spheroids could be propagated long-term. Besides blastemal cultures, we could generate spheroids representing epithelial and stromal WT. Spheroid cultures were analyzed by immunohistochemistry in comparison to corresponding tumor sections and were further characterized by RNA sequencing. Histological appearance of spheroids resembled the original tumor and they expressed marker genes characteristic of early renal development and blastemal WT elements. The cultures were amenable to genetic manipulation and they formed xenograft tumors, which resemble the primary human tumor. This collection of WT spheroids that carry different genetic drivers forms a long-sought tool for drug testing and in vitro modeling.

Soft tissue tumors of infancy encompass an overlapping spectrum of diseases that pose unique diagnostic and clinical challenges. We studied genomes and transcriptomes of cryptogenic congenital mesoblastic nephroma (CMN), and extended our findings to five anatomically or histologically related soft tissue tumors: infantile fibrosarcoma (IFS), nephroblastomatosis, Wilms tumor, malignant rhabdoid tumor, and clear cell sarcoma of the kidney. A key finding is recurrent mutation of EGFR in CMN by internal tandem duplication of the kinase domain, thus delineating CMN from other childhood renal tumors. Furthermore, we identify BRAF intragenic rearrangements in CMN and IFS. Collectively these findings reveal novel diagnostic markers and therapeutic strategies and highlight a prominent role of isolated intragenic rearrangements as drivers of infant tumors.

Internal tandem duplication within the BCOR gene sequence that encodes the PUFD domain, important in the formation of the non-canonical or variant polycomb repressor complex 1 (v-PRC1), was very recently described in 100% of 20 clear cell sarcomas of kidney (CCSKs). None of those 20 cases bore the YWHAE-NUTM2 transcript, previously described by us in CCSK, and which constitutes the only other recurrent genetic aberration observed in CCSK, prompting consideration that these mutations might be mutually exclusive in CCSK. We analysed a cohort of 159 CCSKs and can now not only confirm that there is indeed mutual exclusivity of these BCOR and YWHAE mutations, but also show that a substantial proportion (in this series 11.8%) of CCSKs bear neither mutation when tested by these assays, raising the possibility of distinct aetiologies for subsets of CCSK. Clinical differences observed between the subsets support this notion. As CCSK may show poor chemo-responsiveness, and current treatment protocols mandate the use of doxorubicin with its associated side-effects, advances in understanding the disease biology with a view to more targeted and personalized treatment is a pressing need.

SummaryBlastemal histology in chemotherapy-treated pediatric Wilms tumors (nephroblastoma) is associated with adverse prognosis. To uncover the underlying tumor biology and find therapeutic leads for this subgroup, we analyzed 58 blastemal type Wilms tumors by exome and transcriptome sequencing and validated our findings in a large replication cohort. Recurrent mutations included a hotspot mutation (Q177R) in the homeo-domain of SIX1 and SIX2 in tumors with high proliferative potential (18.1% of blastemal cases); mutations in the DROSHA/DGCR8 microprocessor genes (18.2% of blastemal cases); mutations in DICER1 and DIS3L2; and alterations in IGF2, MYCN, and TP53, the latter being strongly associated with dismal outcome. DROSHA and DGCR8 mutations strongly altered miRNA expression patterns in tumors, which was functionally validated in cell lines expressing mutant DROSHA.

Functional analysis of gene candidates and testing of novel therapeutics in Wilms tumors (WT) has been hampered by the lack of in vitro model systems. WT are characterized by a spectrum of histological appearances, but published cell lines are mostly derived from rare anaplastic variants or even non-WT. There has been some success in establishing primary cultures, but these are often poorly characterized or only derived from less frequent WT1 mutant tumors. We report the generation of a set of primary WT-cell cultures using a simple cultivation protocol. Our cultures could be established after preoperative chemotherapy and irrespective of histological subtypes or genetic alterations. The presence of tumor-specific genetic alterations validates these cultures as being tumor-derived. Genetic characterization is of utmost importance as some cultures with similar morphological appearance lacked such alterations and either represent clonal variants or normal cells. By immunohistochemistry, the cells are either epithelial or more mesenchymal, and the latter exhibiting a longer life span with 30 or more passages before undergoing senescence. This may be related to WT being embryonal tumors with a strong differentiation potential that may prevail in vitro. Telomeres progressively shorten with cultivation, but their length does not predict lifespan. hTERT transfection may partly allow establishment of immortalized lines, because 2/7 cultures avoid senescence even in later passages. Importantly, these cells can be efficiently manipulated by transfection, making them a useful model system for in vitro testing.

Internal tandem duplication within the BCOR gene sequence that encodes the PUFD domain, important in the formation of the non-canonical or variant polycomb repressor complex 1 (v-PRC1), was very recently described in 100% of 20 clear cell sarcomas of kidney (CCSKs). None of those 20 cases bore the YWHAE-NUTM2 transcript, previously described by us in CCSK, and which constitutes the only other recurrent genetic aberration observed in CCSK, prompting consideration that these mutations might be mutually exclusive in CCSK. We analysed a cohort of 159 CCSKs and can now not only confirm that there is indeed mutual exclusivity of these BCOR and YWHAE mutations, but also show that a substantial proportion (in this series 11.8%) of CCSKs bear neither mutation when tested by these assays, raising the possibility of distinct aetiologies for subsets of CCSK. Clinical differences observed between the subsets support this notion. As CCSK may show poor chemo-responsiveness, and current treatment protocols mandate the use of doxorubicin with its associated side-effects, advances in understanding the disease biology with a view to more targeted and personalized treatment is a pressing need.