Generation of a Wt1 conditional deletion, nuclear red fluorescent protein reporter allele in the mouse.

A Wt1 conditional deletion, nuclear red fluorescent protein (RFP) reporter allele was generated in the mouse by gene targeting in embryonic stem cells. Upon Cre-mediated recombination, a deletion allele is generated that expresses RFP in a Wt1-specific pattern. RFP expression was detected in embryonic and adult tissues known to express Wt1, including the kidney, mesonephros, and testis. In addition, RFP expression and WT1 co-localization was detected in the adult uterine stroma and myometrium, suggesting a role in uterine function. Crosses with Wnt7a-Cre transgenic mice that express Cre in the Müllerian duct epithelium activate Wt1-directed RFP expression in the epithelium of the oviduct but not the stroma and myometrium of the uterus. This new mouse strain should be a useful resource for studies of Wt1 function and marking Wt1-expressing cells.

Role of Cyclooxygenase-2 Pathway in Creating an Immunosuppressive Microenvironment and in Initiation and Progression of Wilms' Tumor.

Wilms' tumors (WT), which accountfor 6% of all childhood cancers, arise from dysregulated differentiation of nephrogenic progenitor cells from embryonic kidneys. Though there is an improvement in the prognosis of WT, still 10% of patients with WT die due to recurrence. Thus more effective treatment approaches are necessary. We previously characterized the inflammatory microenvironment in human WT and observed the robust expression of COX-2. The aim of this study was to extend our studies to analyze the role of COX-2 pathway components in WT progression using a mouse model of WT. Herein, COX-2 pathway components such as COX-2, HIF1-α, p-ERK1/2, and p-STAT3 were upregulated in mouse and human tumor tissues. In our RPPA analysis, COX-2 was up-regulated in M15 cells after Wt1 gene was knocked down. Flow cytometry analysis showed the increased infiltration of immune suppressive inflammatory cells such as pDC's and Treg cells in tumors. The chemotactic chemokines responsible for the infiltration of these cells were also induced in CCR5 and CXCR4 dependent manner respectively. The immunosuppressive cytokines IL-10, TGF-ß, and TNF-α were also up-regulated. Furthermore, more pronounced Th2 and Treg induced cytokine response was observed than Th1 response in tumors. Basing on all these evidences it is speculated that COX-2 pathway may be a beneficial target for the treatment of WT. It may be most effective as an adjuvant therapy together with other inhibitors. Thus, our current study provides a good rationale for initiating animal studies to confirm the efficacy of COX-2 inhibitors in decreasing tumor cell growth in vivo.

Nephron Progenitor But Not Stromal Progenitor Cells Give Rise to Wilms Tumors in Mouse Models with ß-Catenin Activation or Wt1 Ablation and Igf2 Upregulation.

Wilms tumor, a common childhood tumor of the kidney, is thought to arise from undifferentiated renal mesenchyme. Variable tumor histology and the identification of tumor subsets displaying different gene expression profiles suggest that tumors may arise at different stages of mesenchyme differentiation and that this ontogenic variability impacts tumor pathology, biology, and clinical outcome. To test the tumorigenic potential of different cell types in the developing kidney, we used kidney progenitor-specific Cre recombinase alleles to introduce Wt1 and Ctnnb1 mutations, two alterations observed in Wilms tumor, into embryonic mouse kidney, with and without biallelic Igf2 expression, another alteration that is observed in a majority of tumors. Use of a Cre allele that targets nephron progenitors to introduce a Ctnnb1 mutation that stabilizes ß-catenin resulted in the development of tumors with a predominant epithelial histology and a gene expression profile in which genes characteristic of early renal mesenchyme were not expressed. Nephron progenitors with Wt1 ablation and Igf2 biallelic expression were also tumorigenic but displayed a more triphasic histology and expressed early metanephric mesenchyme genes. In contrast, the targeting of these genetic alterations to stromal progenitors did not result in tumors. These data demonstrate that committed nephron progenitors can give rise to Wilms tumors and that committed stromal progenitors are less tumorigenic, suggesting that human Wilms tumors that display a predominantly stromal histology arise from mesenchyme before commitment to a stromal lineage.

Identification of germline DICER1 mutations and loss of heterozygosity in familial Wilms tumour.

Wilms tumour (WT), a paediatric renal cancer, is the most common childhood kidney cancer. The aetiology of WT is heterogeneous with multiple genes known to result in WT tumorigenesis. However, these genes are rarely associated with familial Wilms tumour (FWT). To identify mutations predisposing to FWT, we performed whole-genome sequencing using genomic DNA from three affected/obligate carriers in a large WT family, followed by Sanger sequencing of candidate gene mutations in 47 additional WT families to determine their frequency in FWT. As a result, we identified two novel germline DICER1 mutations (G803R and R800Xfs5) co-segregating in two families, thus expanding the number of reported WT families with unique germline DICER1 mutations. The one large family was found to include individuals with multiple DICER1 syndrome phenotypes, including four WT cases. Interestingly, carriers of the DICER1 mutation displayed a greatly increased frequency of WT development compared with the penetrance observed in previously published pedigrees. Also uniquely, in one tumour this DICER1 mutant allele (G803R) was reduced to homozygosity in contrast to the somatic hotspot mutations typically observed in tumours in DICER1 families.

ß-catenin activation in a novel liver progenitor cell type is sufficient to cause hepatocellular carcinoma and hepatoblastoma.

Hepatocellular carcinoma (HCC) was thought historically to arise from hepatocytes, but gene expression studies have suggested that it can also arise from fetal progenitor cells or their adult progenitor progeny. Here, we report the identification of a unique population of fetal liver progenitor cells in mice that can serve as a cell of origin in HCC development. In the transgenic model used, mice carry the Cited1-CreER(TM)-GFP BAC transgene in which a tamoxifen-inducible Cre (CreER(TM)) and GFP are controlled by a 190-kb 5' genomic region of Cited1, a transcriptional coactivator protein for CBP/p300. Wnt signaling is critical for regulating self-renewal of progenitor/stem cells and has been implicated in the etiology of cancers of rapidly self-renewing tissues, so we hypothesized that Wnt pathway activation in CreER(TM)-GFP(+) progenitors would result in HCC. In livers from the mouse model, transgene-expressing cells represented 4% of liver cells at E11.5 when other markers were expressed, characteristic of the hepatic stem/progenitor cells that give rise to adult hepatocytes, cholangiocytes, and SOX9(+) periductal cells. By 26 weeks of age, more than 90% of Cited1-CreER(TM)-GFP;Ctnnb1(ex3(fl)) mice with Wnt pathway activation developed HCC and, in some cases, hepatoblastomas and lung metastases. HCC and hepatoblastomas resembled their human counterparts histologically, showing activation of Wnt, Ras/Raf/MAPK, and PI3K/AKT/mTOR pathways and expressing relevant stem/progenitor cell markers. Our results show that Wnt pathway activation is sufficient for malignant transformation of these unique liver progenitor cells, offering functional support for a fetal/adult progenitor origin of some human HCC. We believe this model may offer a valuable new tool to improve understanding of the cellular etiology and biology of HCC and hepatoblastomas and the development of improved therapeutics for these diseases.

Rare variant detection using family-based sequencing analysis.

Next-generation sequencing is revolutionizing genomic analysis, but this analysis can be compromised by high rates of missing true variants. To develop a robust statistical method capable of identifying variants that would otherwise not be called, we conducted sequence data simulations and both whole-genome and targeted sequencing data analysis of 28 families. Our method (Family-Based Sequencing Program, FamSeq) integrates Mendelian transmission information and raw sequencing reads. Sequence analysis using FamSeq reduced the number of false negative variants by 14-33% as assessed by HapMap sample genotype confirmation. In a large family affected with Wilms tumor, 84% of variants uniquely identified by FamSeq were confirmed by Sanger sequencing. In children with early-onset neurodevelopmental disorders from 26 families, de novo variant calls in disease candidate genes were corrected by FamSeq as mendelian variants, and the number of uniquely identified variants in affected individuals increased proportionally as additional family members were included in the analysis. To gain insight into maximizing variant detection, we studied factors impacting actual improvements of family-based calling, including pedigree structure, allele frequency (common vs. rare variants), prior settings of minor allele frequency, sequence signal-to-noise ratio, and coverage depth (∼20× to >200×). These data will help guide the design, analysis, and interpretation of family-based sequencing studies to improve the ability to identify new disease-associated genes.

Clinically relevant subsets identified by gene expression patterns support a revised ontogenic model of Wilms tumor: a Children's Oncology Group Study.

Wilms tumors (WT) have provided broad insights into the interface between development and tumorigenesis. Further understanding is confounded by their genetic, histologic, and clinical heterogeneity, the basis of which remains largely unknown. We evaluated 224 WT for global gene expression patterns; WT1, CTNNB1, and WTX mutation; and 11p15 copy number and methylation patterns. Five subsets were identified showing distinct differences in their pathologic and clinical features: these findings were validated in 100 additional WT. The gene expression pattern of each subset was compared with published gene expression profiles during normal renal development. A novel subset of epithelial WT in infants lacked WT1, CTNNB1, and WTX mutations and nephrogenic rests and displayed a gene expression pattern of the postinduction nephron, and none recurred. Three subsets were characterized by a low expression of WT1 and intralobar nephrogenic rests. These differed in their frequency of WT1 and CTNNB1 mutations, in their age, in their relapse rate, and in their expression similarities with the intermediate mesoderm versus the metanephric mesenchyme. The largest subset was characterized by biallelic methylation of the imprint control region 1, a gene expression profile of the metanephric mesenchyme, and both interlunar and perilobar nephrogenic rests. These data provide a biologic explanation for the clinical and pathologic heterogeneity seen within WT and enable the future development of subset-specific therapeutic strategies. Further, these data support a revision of the current model of WT ontogeny, which allows for an interplay between the type of initiating event and the developmental stage in which it occurs.

Wt1 ablation and Igf2 upregulation in mice result in Wilms tumors with elevated ERK1/2 phosphorylation.

Wilms tumor (WT) is a genetically heterogeneous childhood kidney tumor. Several genetic alterations have been identified in WT patients, including inactivating mutations in WT1 and loss of heterozygosity or loss of imprinting at 11p15, which results in biallelic expression of IGF2. However, the mechanisms by which one or a combination of genetic alterations results in tumorigenesis has remained challenging to determine, given the lack of a mouse model of WT. Here, we engineered mice to sustain mosaic, somatic ablation of Wt1 and constitutional Igf2 upregulation, mimicking a subset of human tumors. Mice with this combination of genetic alterations developed tumors at an early age. Mechanistically, Wt1 ablation blocked mesenchyme differentiation, and increased Igf2 expression upregulated ERK1/2 phosphorylation. Importantly, a subset of human tumors similarly displayed upregulation of ERK1/2 phosphorylation, which suggests ERK signaling might contribute to WT development. Thus, we have generated a biologically relevant mouse model of WT and defined one combination of driver alterations for WT. This mouse model will provide a powerful tool to study the biology of WT initiation and progression and to investigate therapeutic strategies for cancers with IGF pathway dysregulation.

WT1 mutation and 11P15 loss of heterozygosity predict relapse in very low-risk wilms tumors treated with surgery alone: a children's oncology group study.

PURPOSE: Children's Oncology Group defines very low-risk Wilms tumors (VLRWT) as stage I favorable histology Wilms tumors weighing less than 550 g in children younger than 24 months of age. VLRWTs may be treated with nephrectomy alone. However, 10% to 15% of VLRWTs relapse without chemotherapy. Previous studies suggest that VLRWTs with low WT1 expression and/or 11p15 loss of heterozygosity (LOH) may have increased risk of relapse. The current study validates these findings within prospectively identified children with VLRWT who did not receive adjuvant chemotherapy. PATIENTS AND METHODS: Fifty-six VLRWTs (10 relapses) were analyzed for mutation of WT1, CTNNB1, and WTX; for 11p15 LOH using microsatellite analysis; and for H19DMR and KvDMR1 methylation. RESULTS: 11p15 LOH was identified in 19 (41%) of 46 evaluable VLRWTs and was significantly associated with relapse (P < .001); 16 of 19 were isodisomic for 11p15. WT1 mutation was identified in nine (20%) of 45 evaluable VLRWTs and was significantly associated with relapse (P = .004); all nine cases also had 11p15 LOH. All evaluable tumors showing LOH by microsatellite analysis also showed LOH by methylation analysis. Retention of the normal imprinting pattern was identified in 24 of 42 evaluable tumors, and none relapsed. Loss of imprinting at 11p15 was identified in one of 42 tumors. CONCLUSION: WT1 mutation and 11p15 LOH are associated with relapse in patients with VLRWTs who do not receive chemotherapy. These may provide meaningful biomarkers to stratify patients for reduced chemotherapy in the future. VLRWTs show a different incidence of WT1 mutation and 11p15 imprinting patterns than has been reported in Wilms tumors of all ages.

Loss of heterozygosity at 2q37 in sporadic Wilms' tumor: putative role for miR-562.

PURPOSE: Wilms' tumor is a childhood cancer of the kidney with an incidence of approximately 1 in 10,000. Cooccurrence of Wilms' tumor with 2q37 deletion syndrome, an uncommon constitutional chromosome abnormality, has been reported previously in three children. Given these are independently rare clinical entities, we hypothesized that 2q37 harbors a tumor suppressor gene important in Wilms' tumor pathogenesis. EXPERIMENTAL DESIGN: To test this, we performed loss of heterozygosity analysis in a panel of 226 sporadic Wilms' tumor samples and mutation analysis of candidate genes. RESULTS: Loss of heterozygosity was present in at least 4% of cases. Two tumors harbored homozygous deletions at 2q37.1, supporting the presence of a tumor suppressor gene that follows a classic two-hit model. However, no other evidence of second mutations was found, suggesting that heterozygous deletion alone may be sufficient to promote tumorigenesis in concert with other genomic abnormalities. We show that miR-562, a microRNA within the candidate region, is expressed only in kidney and colon and regulates EYA1, a critical gene for renal development. miR-562 expression is reduced in Wilms' tumor and may contribute to tumorigenesis by deregulating EYA1. Two other candidate regions were localized at 2q37.3 and 2qter, but available data from patients with constitutional deletions suggest that these probably do not confer a high risk for Wilms' tumor. CONCLUSIONS: Our data support the presence of a tumor suppressor gene at 2q37.1 and suggest that, in individuals with constitutional 2q37 deletions, any increased risk for developing Wilms' tumor likely correlates with deletions encompassing 2q37.1.

Wilms tumor genetics: mutations in WT1, WTX, and CTNNB1 account for only about one-third of tumors.

Wilms tumor is genetically heterogeneous, and until recently only one Wilms tumor gene was known, WT1 at 11p13. However, WT1 is altered in only approximately 20% of Wilms tumors. Recently a novel gene, WTX at Xq11.1, was reported to be mutated in Wilms tumors. No overlap between tumors with mutations in WTX and WT1 was noted, suggesting that WT1 and WTX mutations could account for the genetic basis of roughly half of Wilms tumors. To assess the frequency of WTX mutations and their relationship to WT1 mutations in a larger (n = 125) panel of Wilms tumors which had been thoroughly assessed for mutations in WT1, we conducted a complete mutational analysis of WTX that included sequencing of the entire coding region and quantitative PCR to identify deletions of the WTX gene. Twenty-three (18.4%) tumors carried a total of 24 WTX mutations, a lower WTX mutation frequency than that previously observed. Surprisingly, we observed an equivalent frequency of WTX mutations in tumors with mutations in either or both WT1 and CTNNB1 (20.0%) and tumors with no mutation in either WT1 or CTNNB1 (17.5%). WTX has been reported to play a role in the WNT/beta-catenin signaling pathway, and, interestingly, WTX deletion/truncation mutations appeared to be rare in tumors carrying exon 3 mutations of CTNNB1, encoding beta-catenin. Our findings indicate that WT1 and WTX mutations occur with similar frequency, that they partially overlap in Wilms tumors, and that mutations in WT1, WTX, and CTNNB1 underlie the genetic basis of about one-third of Wilms tumors.

Genome-wide loss of heterozygosity analysis of WT1-wild-type and WT1-mutant Wilms tumors.

Wilms tumor (WT) is genetically heterogeneous, and the one known WT gene, WT1 at 11p13, is altered in only a subset of WTs. Previous loss of heterozygosity (LOH) analyses have revealed the existence of additional putative WT genes at 11p15, 16q, and 1p, but these analyses examined only one or a handful of chromosomes or looked at LOH at only a few markers per chromosome. We conducted a genome-wide scan for LOH in WT by using 420 markers spaced at an average of 10 cM throughout the genome and analyzed the data for two genetically defined subsets of WTs: those with mutations in WT1 and those with no detectable WT1 alteration. Our findings indicated that the incidence of LOH throughout the genome was significantly lower in our group of WTs with WT1 mutations. In WT1-wild-type tumors, we observed the expected LOH at 11p, 16q, and 1p, and, in addition, we localized a previously unobserved region of LOH at 9q. Using additional 9q markers within this region of interest, we sublocalized the region of 9q LOH to the 12.2 Mb between D9S283 and a simple tandem repeat in BAC RP11-177I8, a region containing several potential tumor-suppressor genes. As a result, we have established for the first time that WT1-mutant and WT1-wild-type WTs differ significantly in their patterns of LOH throughout the genome, suggesting that the genomic regions showing LOH in WT1-wild-type tumors harbor genes whose expression is regulated by the pleiotropic effects of WT1. Our results implicate 9q22.2-q31.1 as a region containing such a gene.

Familial Wilms tumor.

Wilms tumor (WT), an embryonic tumor arising from undifferentiated renal mesenchyme, has been a productive model for understanding the role of genes in both tumorigenesis and normal organogenesis. Approximately 2% of WT patients have a family history of WT, and even sporadic WT is thought to have a strong genetic component to its etiology. Familial WT cases generally have an earlier age of onset and an increased frequency of bilateral disease, although there is variability among WT families, with some families displaying later than average ages at diagnosis. One WT gene, WT1 at 11p13, has been cloned, but only a minority of tumors carry detectable mutations at that locus, and it can be excluded as the predisposition gene in most WT families. Two familial WT genes have been localized, FWT1 at 17q12-q21 and FWT2 at 19q13.4; lack of linkage in some WT families to either of these loci implies the existence of at least one additional familial WT gene. Originally modeled as the inheritance of a mutation in a tumor suppressor gene, molecular analysis of familial tumors not linked to 11p13 have provided data suggesting that this model may be overly simplistic and/or not applicable to all WT families. Identification of the FWT1 and FWT2 genes will help clarify this and will also likely aid in our understanding in general of the roles of the various WT genes and their genetic interactions in the development of WT.