New mutation in WT1 gene in a boy with an incomplete form of Denys-Drash syndrome: A CARE-compliant case report.

RATIONALE: Pediatric patients with WTl-associated syndromes (including Wilms' tumor-aniridia syndrome and Denys-Drash syndrome), Perlman syndrome, mosaic aneuploidy, and Fanconi anemia with a biallelic breast cancer type 2 susceptibility protein mutation have the highest risk of developing Wilms' tumor. PATIENT CONCERNS AND DIAGNOSIS: We describe a patient with bilateral metachronous Wilms' tumor, ambiguous genitalia characterized by 46, XY disorder of sexual development (DSD) with scrotal hypospadias and bilateral abdominal cryptorchidism, but without nephropathy. At the age of 7 months, the child underwent left nephrectomy with left orchiopexy. At follow-up after 8 months, a second tumor with a diameter of 10 mm was detected in abdominal CT scans at the lower pole of the right kidney. INTERVENTION: Intra-operative macroscopic inspection of the right kidney revealed a tight attachment of the right proximal ureter to the tumor. Thus, retroperitoneoscopic resection of the lower pole of the right kidney had to be changed to an open surgical procedure with partial resection of the proximal ureter and high uretero-ureterostomy. We subsequently performed orchiopexy and two-stage correction of hypospadias using a free skin graft. OUTCOMES: At the last follow-up at the age of 8 years, no pathology requiring treatment was noted. A pair-end-reading (2 × 125) DNA analysis with an average coverage of at least 70 to 100 × revealed a previously unknown heterozygous mutation in exon 7 of the Wilms' tumor suppressor gene 1 (WT1) gene (chr11:32417947G>A), leading to the appearance of a site of premature translation termination in codon 369 (p.Arg369Ter, NM_024426.4). This mutation had not been registered previously in the control samples "1000 genomes," Exome Sequencing Project 6500, and the Exome Aggregation Consortium. Thus, to the best of our knowledge this represents a newly identified mutation causing incomplete Denys-Drash syndrome.

TRIM28 haploinsufficiency predisposes to Wilms tumor.

Two percent of patients with Wilms tumors have a positive family history. In many of these cases the genetic cause remains unresolved. By applying germline exome sequencing in two families with two affected individuals with Wilms tumors, we identified truncating mutations in TRIM28. Subsequent mutational screening of germline and tumor DNA of 269 children affected by Wilms tumor was performed, and revealed seven additional individuals with germline truncating mutations, and one individual with a somatic truncating mutation in TRIM28. TRIM28 encodes a complex scaffold protein involved in many different processes, including gene silencing, DNA repair and maintenance of genomic integrity. Expression studies on mRNA and protein level showed reduction of TRIM28, confirming a loss-of-function effect of the mutations identified. The tumors showed an epithelial-type histology that stained negative for TRIM28 by immunohistochemistry. The tumors were bilateral in six patients, and 10/11 tumors are accompanied by perilobar nephrogenic rests. Exome sequencing on eight tumor DNA samples from six individuals showed loss-of-heterozygosity (LOH) of the TRIM28-locus by mitotic recombination in seven tumors, suggesting that TRIM28 functions as a tumor suppressor gene in Wilms tumor development. Additionally, the tumors showed very few mutations in known Wilms tumor driver genes, suggesting that loss of TRIM28 is the main driver of tumorigenesis. In conclusion, we identified heterozygous germline truncating mutations in TRIM28 in 11 children with mainly epithelial-type Wilms tumors, which become homozygous in tumor tissue. These data establish TRIM28 as a novel Wilms tumor predisposition gene, acting as a tumor suppressor gene by LOH.

WT1 expression as a potential biomarker of malignancy in canine breast tumor / La expresión de WT1 como un biomarcador potencial maligno en un tumor de mama canino

SUMMARY: Veterinary oncology is very important nowadays to get a better understanding of human carcinogenesis. Estrogen receptor, progesterone receptor and Human Epidermal Growth Factor receptor 2 are frequently evaluated by immunohistochemistry (HIC) in human breast tumor. WT1 is an oncogene, its overexpression has been detected in leukemia and diverse solid tumors like breast cancer, lung cancer and mesothelioma in humans. WT1 expression was evaluated in 15 canine breast tumors (CBT) diagnosed by histopathological analysis to find its relationship with neoplasia and malignancy. IHC and RT-PCR were performed in CBT tissues. Fisher´s test was used to analyze WT1 relationship with malignancy. Of the 15 tumors, 9 (60 %) were diagnosed as benign and 6 (40 %) were malignant. With IHC, WT1 expression was positive only in biopsies diagnosed as malignant. Expression of WT1 by RT-PCR was detected in 14 of the 15 tumors (93.33 %) as well as in control healthy mammary gland. Clinical significance: This study describes for the first time a close correlation between CBT and a positive result for WT1 expression with IHC; hence, it can be used as a biomarker for this neoplasia and as an indicator of malignancy. RT-PCR analysis also showed to be good option to detect WT1 expression. These results will be useful to further investigations to elucidate WT1-related signaling pathways in CBT. Also to know molecules that regulate the translation of this protein as a marker for tumor progression.

RESUMEN: La oncología veterinaria es muy importante hoy en día para comprender mejor la carcinogénesis humana. El receptor de estrógeno, el receptor de progesterona y el receptor 2 del factor de crecimiento epidérmico humano se evalúan con frecuencia mediante inmunohistoquímica (HIC) en tumores de mama humanos. WT1 es un oncogén, su sobreexpresión se ha detectado en leucemia y en diversos tumores sólidos como el cáncer de mama, cáncer de pulmón y mesotelioma en humanos. La expresión de WT1 se evaluó en 15 tumores de mama caninos (TCC) diagnosticados mediante análisis histopatológico para encontrar su relación con la neoplasia y la malignidad. IHC y RT-PCR se realizaron en tejidos CBT. La prueba de Fisher se utilizó para analizar la relación de WT1 con la malignidad. De los 15 tumores, 9 (60 %) fueron diagnosticados como benignos y 6 (40 %) fueron malignos. Con IHC, la expresión de WT1 fue positiva solo en biopsias diagnosticadas como malignas. La expresión de WT1 por RT-PCR se detectó en 14 de los 15 tumores (93,33 %), así como en el control de la glándula mamaria sana. Importancia clínica: este estudio describe por primera vez una estrecha correlación entre la TCC y un resultado positivo para la expresión de WT1 con IHC; por lo tanto, se puede utilizar como un biomarcador para esta neoplasia y como un indicador de malignidad. El análisis por RT-PCR también demostró ser una buena opción para detectar la expresión de WT1. Estos resultados serán útiles para futuras investigaciones para dilucidar las vías de señalización relacionadas con WT1 en la TCC. También para conocer moléculas que regulan la traducción de esta proteína como marcador de progresión tumoral.

Wilms' tumor gene 1 silencing inhibits proliferation of human osteosarcoma MG-63 cell line by cell cycle arrest and apoptosis activation.

Wilms' tumor gene 1 (WT1) plays complex roles in tumorigenesis, acting as tumor suppressor gene or an oncogene depending on the cellular context. A high WT1 expression level was described in various types of human bone and soft-tissue sarcomas, including osteosarcoma (OS), but its function in carcinogenesis is not yet well understood. This study investigated WT1 both in human OS tissues and in human OS MG-63 cell line in which WT1 gene is up-regulated. The results demonstrated that WT1 is expressed in 50% of human OS cases. WT1-silenced MG-63 cells showed deregulation of proteins of cell cycle and down-regulation of PI3K/AKT pathway. Induction of apoptotic programme was also established by activation of caspase-3 and increase of Bax/Bcl2 ratio and p53 protein. This study provided new findings on role of WT1 and indicated an association between WT1 expression, cell cycle and apoptotic machinery. In conclusion, WT1 acts as a tumour promoter in osteosarcoma and it could be a potential therapeutic target.

Prolonging nephrogenesis in preterm infants: a new approach for prevention of kidney disease in adulthood?

Chronic kidney disease represents a dramatic worldwide resource-consuming problem. This problem is of increasing importance even in preterm infants, since nephrogenesis may go on only for a few weeks (4 to 6 weeks) after birth. Recent literature focusing on traditional regenerative medicine does not take into account the presence of a high number of active endogenous stem cells in the preterm kidney, which represents a unique opportunity for starting regenerative medicine in the perinatal period. Pluripotent cells of the blue strip have the capacity to generate new nephrons, improving kidney function in neonates and potentially protecting them from developing chronic kidney disease and end-stage renal disease in adulthood. There is a marked interindividual neonatal variability of nephron numbers. Moreover, the renal stem/progenitor cells appear as densely-packed small cells with scant cytoplasm, giving rise to a blue-appearing strip in hematoxylin-eosin-stained kidney sections ("the blue strip"). There are questions concerning renal regenerative medicine: among preliminary data, the simultaneous expression of Wilms tumor 1 and thymosin ß4 in stem/progenitor cells of the neonatal kidney may bring new prospects for renal regeneration applied to renal stem cells that reside in the kidney itself. A potential approach could be to prolong the 6 weeks of postnatal renal growth of nephrons or to accelerate the growth of nephrons during the 6 weeks or both. Considering what we know today about perinatal programming, this could be an important step for the future to reduce the incidence and global health impact of chronic kidney disease.

Wilms' tumor gene 1 regulates p63 and promotes cell proliferation in squamous cell carcinoma of the head and neck.

BACKGROUND: Wilms' tumor gene 1 (WT1) can act as a suppressor or activator of tumourigenesis in different types of human malignancies. The role of WT1 in squamous cell carcinoma of the head and neck (SCCHN) is not clear. Overexpression of WT1 has been reported in SCCHN, suggesting a possible oncogenic role for WT1. In the present study we aimed at investigating the function of WT1 and its previously identified protein partners p63 and p53 in the SCCHN cell line FaDu. METHODS: Silencing RNA (siRNA) technology was applied to knockdown of WT1, p63 and p53 in FaDu cells. Cell proliferation was detected using MTT assay. Chromatin immunoprecipitation (ChIP)/PCR analysis was performed to confirm the effect of WT1 on the p63 promoter. Protein co-immunoprecipitation (co-IP) was used to find protein interaction between WT1 and p53/p63. Microarray analysis was used to identify changes of gene expression in response to knockdown of either WT1 or p63. WT1 RNA level was detected using real-time quantitative PCR (RT-qPCR) in patients with SCCHN. RESULTS: We found that WT1 and p63 promoted cell proliferation, while mutant p53 (R248L) possessed the ability to suppress cell proliferation. We reported a novel positive correlation between WT1 and p63 expression. Subsequently, p63 was identified as a WT1 target gene. Furthermore, expression of 18 genes involved in cell proliferation, cell cycle regulation and DNA replication was significantly altered by downregulation of WT1 and p63 expression. Several known WT1 and p63 target genes were affected by WT1 knockdown. Protein interaction was demonstrated between WT1 and p53 but not between WT1 and p63. Additionally, high WT1 mRNA levels were detected in SCCHN patient samples. CONCLUSIONS: Our findings suggest that WT1 and p63 act as oncogenes in SCCHN, affecting multiple genes involved in cancer cell growth.

Integration of Cistromic and Transcriptomic Analyses Identifies Nphs2, Mafb, and Magi2 as Wilms' Tumor 1 Target Genes in Podocyte Differentiation and Maintenance.

The Wilms' tumor suppressor gene 1 (WT1) encodes a zinc finger transcription factor. Mutation of WT1 in humans leads to Wilms' tumor, a pediatric kidney tumor, or other kidney diseases, such as Denys-Drash and Frasier syndromes. We showed previously that inactivation of WT1 in podocytes of adult mice results in proteinuria, foot process effacement, and glomerulosclerosis. However, the WT1-dependent transcriptional network regulating podocyte development and maintenance in vivo remains unknown. Here, we performed chromatin immunoprecipitation followed by high-throughput sequencing with glomeruli from wild-type mice. Additionally, we performed a cDNA microarray screen on an inducible podocyte-specific WT1 knockout mouse model. By integration of cistromic and transcriptomic analyses, we identified the WT1 targetome in mature podocytes. To further analyze the function and targets of WT1 in podocyte maturation, we used an Nphs2-Cre model, in which WT1 is deleted during podocyte differentiation. These mice display anuria and kidney hemorrhage and die within 24 hours after birth. To address the evolutionary conservation of WT1 targets, we performed functional assays using zebrafish as a model and identified Nphs2, Mafb, and Magi2 as novel WT1 target genes required for podocyte development. Our data also show that both Mafb and Magi2 are required for normal development of the embryonic zebrafish kidney. Collectively, our work provides insights into the transcriptional networks controlled by WT1 and identifies novel WT1 target genes that mediate the function of WT1 in podocyte differentiation and maintenance.

Wilms' tumor gene WT1 promotes homologous recombination-mediated DNA damage repair.

The Wilms' tumor gene WT1 is overexpressed in leukemia and various types of solid tumors and plays an oncogenic role in these malignancies. Alternative splicing at two sites yields four major isoforms, 17AA(+)KTS(+), 17AA(+)KTS(-), 17AA(-)KTS(+), and 17AA(-)KTS(-), and all the isoforms are expressed in the malignancies. However, among the four isoforms, function of WT1[17AA(-)KTS(+)] isoform still remains undetermined. In the present study, we showed that forced expression of WT1[17AA(-)KTS(+)] isoform significantly inhibited apoptosis by DNA-damaging agents such as Doxorubicin, Mitomycin, Camptothesisn, and Bleomycin in immortalized fibroblast MRC5SV and cervical cancer HeLa cells. Knockdown of Rad51, an essential factor for homologous recombination (HR)-mediated DNA repair canceled the resistance to Doxorubicin induced by WT1[17AA(-)KTS(+)] isoform. GFP recombination assay showed that WT1[17AA(-)KTS(+)] isoform alone promoted HR, but that three other WT1 isoforms did not. WT1[17AA(-)KTS(+)] isoform significantly upregulated the expression of HR genes, XRCC2, Rad51D, and Rad54. Knockdown of XRCC2, Rad51D, and Rad54 inhibited the HR activity and canceled resistance to Doxorubicin in MRC5SV cells with forced expression of WT1[17AA(-)KTS(+)] isoform. Furthermore, chromatin immunoprecipitation (ChIP) assay showed the binding of WT1[17AA(-)KTS(+)] isoform protein to promoters of XRCC2 and Rad51D. Immunohistochemical study showed that Rad54 and XRCC2 proteins were highly expressed in the majority of non-small-cell lung cancer (NSCLC) and gastric cancer, and that expression of these two proteins was significantly correlated with that of WT1 protein in NSCLCs. Our results presented here showed that WT1[17AA(-)KTS(+)] isoform had a function to promote HR-mediated DNA repair.

WT1 expression in endocrine mucin-producing sweat gland carcinoma: a study of 13 cases.

BACKGROUND: Endocrine mucin-producing sweat gland carcinoma (EMPSGC), a low-grade sweat gland carcinoma with a predilection for the eyelids, often shows areas of benign eccrine cysts, atypical intracystic proliferation and associated mucinous carcinoma, suggesting tumor progression. Wilms tumor 1 (WT1) protein, a transcription factor, is overexpressed in many tumors and plays a role in oncogenesis. METHODS: A computer-based search for tumors diagnosed between 1989 and 2009 was conducted. Clinical data were obtained from pathology reports and patient records. Biopsies were reviewed for histologic features. Immunostaining was performed for WT1, chromogranin, synaptophysin, estrogen receptor (ER), epithelial membrane antigen (EMA), polyclonal carcinoembryonic antigen (P-CEA), cytokeratin 7 (CK7), cytokeratin 20 (CK20) and MIB-1. RESULTS: Eight women and five men (mean age: 61.2 years; range: 40-77 years) presented with slow-growing eyelid nodules. Cases of EMPSGC were characterized by the presence of dermal nodules with various growth patterns. Adjacent eccrine cysts were present in five patients, atypical epithelial proliferation within the cyst wall in four patients, and an associated mucinous carcinoma in one patient. All tumors were positive for WT1, CK7, ER, P-CEA and EMA and negative for CK20. Tumors were positive for synaptophysin in 12 cases and chromogranin in nine cases. The MIB-1 proliferation index was low in most cases. No WT1 staining was observed in the overlying epidermis, adnexal structures or areas of benign eccrine cyst. WT1 expression was observed in areas of atypical epithelial proliferation, and the neoplastic cells. CONCLUSIONS: The present study shows WT1 expression in the neoplastic epithelial cells of EMPSGC, areas of atypical intraductal proliferations, and mucinous carcinoma. The absence of WT1 expression in areas of benign eccrine cyst and cutaneous sweat glands suggests WT1 upregulation plays a role in tumor cell proliferation and progression of EMPSGC.

Predictive role of minimal residual disease and log clearance in acute myeloid leukemia: a comparison between multiparameter flow cytometry and Wilm's tumor 1 levels.

In acute myeloid leukemia (AML), the detection of minimal residual disease (MRD) as well as the degree of log clearance similarly identifies patients with poor prognosis. No comparison was provided between the two approaches in order to identify the best one to monitor follow-up patients. In this study, MRD and clearance were assessed by both multiparameter flow cytometry (MFC) and WT1 expression at different time points on 45 AML patients achieving complete remission. Our results by WT1 expression showed that log clearance lower than 1.96 after induction predicted the recurrence better than MRD higher than 77.0 copies WT1/10(4) ABL. Conversely, on MFC, MRD higher than 0.2 % after consolidation was more predictive than log clearance below 2.64. At univariate and multivariate analysis, positive MRD values and log clearance below the optimal cutoffs were associated with a shorter disease-free survival (DFS). At the univariate analysis, positive MRD values were also associated with overall survival (OS). Therefore, post-induction log clearance by WT1 and post-consolidation MRD by MFC represented the most informative approaches to identify the relapse. At the optimal timing of assessment, positive MRD and log-clearance values lower than calculated thresholds similarly predicted an adverse prognosis in AML.

Wilms' tumor 1 gene modulates Fas-related death signals and anti-apoptotic functions in hepatocellular carcinoma.

BACKGROUND: The Wilms' tumor 1 (WT1) gene is known to be overexpressed in hepatocellular carcinoma (HCC) and to upregulate tumor growth and oncogenic potential, although the detailed mechanisms remain to be elucidated. METHODS: We identified host genes involved in WT1 gene modulation of human liver cancer cell lines in vitro, and further characterized genes related to apoptosis. Moreover, we evaluated the alteration of genes by WT1 in 40 HCC and 58 non-HCC human liver samples collected at resection. RESULTS: Analysis of the effect of small interfering RNAs-mediated knock-down of WT1 on apoptosis using an annexin V labeling assay, and on modulation of the activity of caspases-3, -8 and -9, indicated that WT1 has an anti-apoptotic role. We identified three apoptosis-related genes that were modulated by WT1; the cellular FLICE-inhibitory proteins (cFLIP) gene was upregulated, and Fas-associated death domain (FADD) and nuclear factor kappa B (NF-κB) were downregulated. Interestingly, knock-down of FADD or NF-κB resulted in the upregulation of WT1, and the expression of cFLIP changed in parallel with WT1 expression. We further evaluated WT1-mediated alteration of genes in HCC and non-HCC human liver samples. Both HCC and non-HCC tissues that expressed relatively high levels of WT1 showed cFLIP overexpression. CONCLUSIONS: WT1 modulates cFLIP, FADD and NF-κB, and has an anti-apoptotic role in HCC. This mechanism of action of WT1 could be related to the tumor growth and oncogenic potential of HCC.

Pautas en el diagnóstico morfológico, inmunohistoquímico y genético de los tumores de células redondas y pequeñas con especial referencia al sarcoma de Ewing/PNET / The morphological, immunohistochemical and genetic features of Ewing/PNET and their differential diagnosis with other small round cell tumours

El objetivo del presente trabajo es realizar una revisión y actualización donde se describe el espectro de hallazgos morfológicos inmunohistoquímicos (IHQ) y genéticos moleculares en los sarcomas de Ewing/PNET (Ewing/PNET) así como su diagnóstico diferencial con otros tumores de células redondas y pequeñas (TCRP) de hueso y partes blandas. La expresión IHQ de CD99, Fli1, HNK-1 y CAV1, unido a las alteraciones genéticas que muestran estos tumores, apoyan el diagnóstico definitivo de esta neoplasia. La expresión de marcadores epiteliales (CK, EMA y/o CEA) en un TCRP no excluye la posibilidad de un Ewing/PNET; sin embargo, la diferenciación miogénica prácticamente excluye la posibilidad de un Ewing/PNET. La variante atípica de Ewing/PNET frecuentemente muestra solapamiento morfológico e IHQ con otros TCRP, por lo que en estos casos es preciso realizar una confirmación genética para un diagnóstico definitivo. El método más eficiente para llegar a un diagnóstico concluyente así como a un diagnóstico diferencial en este tipo de neoplasias consiste en la integración de los hallazgos clínicos, morfológicos, IHQ y genético-moleculares(AU)

The aim of the present study is to review and describe the spectrum of morphological, immunohistochemical (IHC) and molecular genetic characteristics in Ewing's sarcoma family of tumors (ESFT) and their differential diagnostic with other small round cell tumors (SRCTs) of bone and soft tissue. The diagnosis of ESFT has been supported by genetic analysis and IHC profiling, mainly represented by positivity to CD99, Fli1, HNK-1 and CAV1. The epithelial expression including CKs, EMA and/or CEA positivity in SRCTs does not exclude the diagnosis of ESFT. Presence of a myogenic differentiation should rule out the diagnosis of ESFT. The atypical ESFT present IHC features that overlap with other SRCTs, thus genetic studies should be used in order to attain an accurate diagnosis. In conclusion, the integration of clinical, histopathological, IHC and genetic findings is the method of choice for the diagnosis of ESFT, as well as for the differential diagnosis with other SRCTs(AU)

Stratification of Wilms tumor by genetic and epigenetic analysis.

Somatic defects at five loci, WT1, CTNNB1, WTX, TP53 and the imprinted 11p15 region, are implicated in Wilms tumor, the commonest childhood kidney cancer. In this study we analysed all five loci in 120 Wilms tumors. We identified epigenetic 11p15 abnormalities in 69% of tumors, 37% were H19 epimutations and 32% were paternal uniparental disomy (pUPD). We identified mutations of WTX in 32%, CTNNB1 in 15%, WT1 in 12% and TP53 in 5% of tumors. We identified several significant associations: between 11p15 and WTX (P=0.007), between WT1 and CTNNB1 (P less than 0.001), between WT1 and pUPD 11p15 (P=0.01), and a strong negative association between WT1 and H19 epimutation (P less than 0.001). We next used these data to stratify Wilms tumor into three molecular Groups, based on the status at 11p15 and WT1. Group 1 tumors (63%) were defined as 11p15-mutant and WT1-normal; a third also had WTX mutations. Group 2 tumors (13%) were WT1-mutant. They either had 11p15 pUPD or were 11p15-normal. Almost all had CTNNB1 mutations but none had H19 epimutation. Group 3 tumors (25%) were defined as 11p15-normal and WT1-normal and were typically normal at all five loci (P less than 0.001). We also identified a novel clinical association between H19 epimutation and bilateral disease (P less than 0.001). These data provide new insights into the pattern, order, interactions and clinical associations of molecular events in Wilms tumor.

WT1 in disease: shifting the epithelial-mesenchymal balance.

WT1 is a versatile gene that controls transitions between the mesenchymal and epithelial state of cells in a tissue-context dependent manner. As such, WT1 is indispensable for normal development of many organs and tissues. Uncontrolled epithelial to mesenchymal transition (EMT) is a hallmark of a diverse array of pathologies and disturbance of mesenchymal to epithelial transition (MET) has been associated with a number of developmental abnormalities. It is therefore not surprising that WT1 has been linked to many of these. Here we review the role of WT1 in proper control of the mesenchymal-epithelial balance of cells and discuss how far these roles can explain the role of WT1 in a variety of disease states.

LMP1 antagonizes WNT/ß-catenin signalling through inhibition of WTX and promotes nasopharyngeal dysplasia but not tumourigenesis in LMP1(B95-8) transgenic mice.

Latent membrane protein 1 (LMP1) of Epstein-Barr virus (EBV) can induce cell transformation and tumourigenesis, but the mechanism is not understood. Previous studies have suggested that LMP1 acts through up-regulation of cellular proliferation pathways including the Wnt/ß-catenin pathway, in which ß-catenin is the central effector. Increased levels of ß-catenin coupled with a decrease in E-cadherin lead to reduced cell adhesion. This pathway is antagonized by WTX (Wilms' tumour gene on the X chromosome), which can promote the ubiquitination and degradation of ß-catenin. In the present study, we established L2/LMP1B(95 - 8) /EGFP transgenic mice to investigate the in vivo role of LMP1. Down-regulation of WTX and E-cadherin was accompanied by increased expression of ß-catenin in these mice. Even though invasive tumours did not develop, dysplasia was seen in the nasopharynx and oropharynx epithelium of these transgenic mice. Analysis of LMP1(+) , WTX(+) , and LMP1 siRNA silenced HNE-1 cell lines demonstrated that WTX could exert a dominant role in LMP1-mediated WNT/ß-catenin pathway regulation. This study indicates that LMP1 antagonizes the WNT/ß-catenin pathway by inhibiting WTX, and this reduction in WTX is associated with epithelial dysplasia via regulation of the WNT/ß-catenin pathway molecules E-cadherin and ß-catenin. Further studies are required for a better understanding of the relationship between LMP1-mediated antagonization of the WNT/ß-catenin pathway and tumourigenesis.

The role of WT1 gene in neuroblastoma.

BACKGROUND/PURPOSE: The oncogenic properties of the Wilms' tumor gene (WT1) have recently been reported in various malignancies. However, the role of WT1 in pediatric tumors is unclear. To elucidate the role of WT1 in the development of neuroblastoma (NB), we examined the WT1 expression in NB and the effect of WT1 suppression on NB cell proliferation. METHODS: We examined the expression of the WT1 protein in 20 NBs and 5 ganglioneuromas (GNs) by performing immunohistochemical analysis. We determined WT1 messenger RNA expression in 22 NBs, 5 GNs, and 4 NB cell lines by real-time reverse transcription polymerase chain reaction. We studied the effects of WT1 suppression on cell proliferation using small interfering RNA against WT1. RESULTS: Expression of WT1 was higher in mature ganglionic cells, and in the immunohistochemical analysis, the WT1 positivity for GNs was significantly higher than that for NBs (P < .01). The level of WT1 messenger RNA expression did not correlate with histologic grade, clinical stage, and prognosis of the tumor. Knockdown of WT1 gene promoted the proliferation of NB69 cells (P < .01). CONCLUSIONS: The WT1 may govern cell differentiation and suppress cell proliferation in NB. The WT1 does not act as an oncogene, but it may participate in the maturation of NB.

Characterization of mesonephric development and regeneration using transgenic zebrafish.

The zebrafish is a valuable vertebrate model for kidney research. The majority of previous studies focused on the zebrafish pronephros, which comprises only two nephrons and is structurally simpler than the mesonephros of adult fish and the metanephros of mammals. To evaluate the zebrafish system for more complex studies of kidney development and regeneration, we investigated the development and postinjury regeneration of the mesonephros in adult zebrafish. Utilizing two transgenic zebrafish lines (wt1b::GFP and pod::NTR-mCherry), we characterized the developmental stages of individual mesonephric nephrons and the temporal-spatial pattern of mesonephrogenesis. We found that mesonephrogenesis continues throughout the life of zebrafish, with a rapid growth phase during the juvenile period and a slower phase in adulthood such that the total nephron number of juvenile and adult fish linearly correlates with body mass. Following gentamicin-induced renal injury, the zebrafish mesonephros can undergo de novo regeneration of mesonephric nephrons, a process known as neonephrogenesis. We found that wt1b expression was induced in individually dispersed cells in the mesonephric interstitium as early as 48 h following injury. These wt1b-expressing cells formed aggregates by 72-96 h following injury which proceeded to form nephrons. This suggests that wt1b may serve as an early marker of fated renal progenitor cells. The synchronous nature of regenerative neonephrogenesis suggests that this process may be useful for studies of nephron development.

Prognostic implications of mutations and expression of the Wilms tumor 1 (WT1) gene in adult acute T-lymphoblastic leukemia.

BACKGROUND: The role of the Wilms tumor 1 gene (WT1) in acute leukemias has been underscored by mutations found in acute myeloid leukemia identifying patients with inferior survival. Furthermore, aberrant expression of WT1 in acute myeloid leukemia was associated with an increased risk of relapse. No larger studies have performed a combined approach including WT1 mutation and expression analyses in acute T-lymphoblastic leukemia. DESIGN AND METHODS: We analyzed the WT1 mutations and the expression status in a total of 252 consecutive adult patients with newly diagnosed T-lymphoblastic leukemia, who were registered on the GMALL 06/99 and 07/03 protocols and had sufficient material available. The GMALL protocols included intensive chemotherapy as well as stem cell transplantation according to a risk-based model with indication for stem cell transplantation in first complete remission for early and mature T-lymphoblastic leukemia patients; patients with thymic T-lymphoblastic leukemia were allocated to a standard risk group and treated with intensive chemotherapy. RESULTS: Twenty of the 238 patients analyzed had WT1 mutations (WT1mut) in exon 7. WT1mut cases were characterized by immature features such as an early immunophenotype and higher WT1 expression. In thymic T-lymphoblastic leukemia, WT1mut patients had an inferior relapse-free survival compared to WT1 wild-type patients. T-lymphoblastic leukemia patients with aberrant WT1 expression (high or negative) showed a higher relapse rate and an inferior outcome compared to patients with intermediate WT1 expression. In the standard risk group of thymic T-lymphoblastic leukemia, aberrant WT1 expression was predictive for an inferior relapse-free survival as compared to patients with intermediate expression. In multivariate analysis, WT1 expression was of independent prognostic significance for relapse-free survival. CONCLUSIONS: WT1 mutations were associated with an inferior relapse-free survival in standard risk thymic T-lymphoblastic leukemia patients. Moreover, altered expression associated with inferior outcome also suggests a role of WT1 in T-lymphoblastic leukemia and the potential use of molecularly-based treatment stratification to improve outcome.

Adenovirus vaccine immunotherapy targeting WT1-expressing tumors.

IMPORTANCE OF THE FIELD: Tumor associated antigens (TAAs) offer specific targets for developing cancer immunotherapies. In particular, viral vectors encoding transgenic TAAs have been used in recent vaccination strategies. Wilm's Tumor gene (WT1) is a robust TAA which is overexpressed in many malignancies and has been recently used to develop a novel recombinant adenovirus (Ad-WT1) for antitumor immunotherapy. AREAS COVERED IN THIS REVIEW: The lines of evidence over the past two decades leading to the development of Ad-WT1 immunotherapy are reviewed, including preclinical studies and clinical trials using WT1-based vaccines and TAA-expressing adenoviral vectors for antitumor therapy. WHAT THE READER WILL GAIN: The fundamental immunogenic properties of WT1-based vaccines are detailed, as well as the recent progress in using adenoviral vectors for eliciting a TAA-specific immune response. The reader will also gain an understanding of the evidence supporting Ad-WT1 antitumor therapy in vivo. TAKE HOME MESSAGE: Ad-WT1 elicits a potent CD4(+) and CD8(+) T cell immune response and can effectively inhibit tumor growth in vivo, thus making it an important potential cancer therapy worthy of future investigation.