Detection of type 1 insulinlike growth factor (IGF) receptors in Wilms'' tumors.

Insulinlike growth factors (IGFs) are peptide hormones that regulate proliferation and differentiation of several types of normal and neoplastic cells. Recent studies of Wilms' tumor, a childhood kidney neoplasm, have detected increased expression of IGF-2 mRNA and protein. The present report describes detection of Type 1 IGF receptors in specimens of Wilms' tumor and adjacent nonneoplastic kidney tissue. These receptors recognize both IGF-1 and IGF-2, and binding of either peptide activates endogenous tyrosine kinase activity of the Type 1 IGF receptor beta subunit. These data indicate that Wilms' tumors contain receptors that recognize and respond to exogenous IGF in vitro, and that autocrine IGF production might contribute to the increased proliferation and abnormal differentiation of these cells in vivo.     

The in vitro growth and characterization of the skeletal muscle component of Wilms'' tumor.

Skeletal muscle differentiation within a Wilms' tumor is a well-documented histopathologic entity thought to occur at a relatively low incidence and influence prognosis. A serum-free hormonally defined growth medium has been developed, allowing the long-term growth of the skeletal muscle component of Wilms' tumors. Eight Wilms' tumors have been grown under these conditions. Three cases grew a homogeneous population of cells which ultrastructurally displayed all stages of myogenesis through myotubule formation. They also possessed immunoreactivity for skeletal muscle myosin and myoglobin and synthesized the M and B subunits of creatine kinase. Of interest was the finding that the ability to yield skeletal muscle cultures was limited to those cases which exhibited skeletal muscle fibers in vivo. This technique is also a very sensitive marker for identifying Wilms' tumors possessing a myoid component. A second serum-free hormonally defined medium has also been developed that supports the long-term culture of a unique cell type from Wilms' tumors which contain a myoid component. These cells are spindle-shaped and exhibit all of the characteristics of early myoblasts.     

Wilms' tumor: a search for the critical lesion

A Wilms' tumor from a 12-month-old boy showed epithelial and mainly rhabdomyoblastic differentiation. In addition, the kidney contained foci of nephroblastomatosis, a lesion predisposing to the development of nephric tumors. Flow cytometry indicated that the tumor DNA content was in the diploid range with an increased S-phase. Chromosome studies of the cultured tumor cells showed a dominant pattern of 49,XY, +8,9qh+, +12, +12,18q+, without obvious deletion of 11p. A few cells showed additional losses, deletions, or structural rearrangements superimposed on the basic pattern, but no normal metaphases were observed. The DNA from the tumor was probed for several loci on 11p because variations of 11p (deletion or translocation) have been reported in roughly one third of Wilms' tumors, and the critical gene in Wilms' has been localized to 11p13. In this case, 11p genes maintained heterozygosity or showed no detectable alteration in gene dosage when compared with peripheral-blood DNA. Therefore, despite histologic indication of an underlying constitutional defect, no genomic lesion of 11p was identified.     

Wilms' Tumor 1 Susceptibility (WT1) Gene Products are Selectively Expressed in Malignant Mesothelioma

The distinction between malignant mesothelioma and other neoplastic processes involving the pleura is difficult, partly due to the lack of specific markers expressed on mesothelioma. Because of evidence suggesting that the Wilms' tumor susceptibility gene (WT1), unlike ot her tumor suppressor genes, is restricted mostly to mesenchymaly derived tissues, we hypothesized that the WTI gene products could serve as a potential marker for mesothelioma. The expression of WTI mRNA was analyzed in 19 malignant mesothelioma cell lines and 9 tumors and compared with the expression of WT1 in 10 non-small cell lung cancer lines and 9 lung cancer specimens. WTI mRNA was detectable by Northern analysis in 16 of 19 mesothelioma cell lines and in 5 of 8 malignant mesothelioma tumors. In contrast, WTI mRNA was not detected by Northern analysis in non-small cell lung cancer lines or carcinomas. Immunoprecipitation with an anti-WT1 monoclonal antibody showed that a52-to 54-kdprotein was present in 4 mesothelioma cell lines. Immunostanig with this antibody localizd the WT1 protein to the nucleus in two mesothelioma lines and in 20 of 21 mesothelioma tumors examined. This distinctive pattern of nuclear immunoreactivity was absent in 26 non-mesothelioma tumors involving the lung, including 20 non-small cell lung carcinomas. The detection of WTI mRNA or protein may thus provide a specific molecular or immunohistochemical marker for differentiation of mesothelioma from other pleural tumors, inparticular, adenocarcinoma.     

Differential expression of a novel ankyrin containing E3 ubiquitin-protein ligase, Hace1, in sporadic Wilms' tumor versus normal kidney

We have analyzed the chromosome 6q21 breakpoint of a non-constitutional t(6;15)(q21;q21) rearrangement in sporadic Wilms' tumor. This identified a novel gene encoding a protein with six N-terminal ankyrin repeats linked to a C-terminal HECT ubiquitin-protein ligase domain. We therefore designated this gene HACE1 (HECT domain and Ankyrin repeat Containing E3 ubiquitin-protein ligase 1). HACE1 is widely expressed in human tissues, including mature and fetal kidney. We show that Hace1 protein possesses intrinsic ubiquitin ligase activity, utilizes UbcH7 as a candidate partner E2 enzyme and localizes predominantly to the endoplasmic reticulum. Although the HACE1 locus was not directly interrupted by the translocation in the index Wilms' case, its expression was markedly lower in tumor tissue compared with adjacent normal kidney. Moreover, HACE1 expression was virtually undetectable in the SK-NEP-1 Wilms' tumor cell line and in four of five additional primary Wilms' tumor cases compared with patient-matched normal kidney. We found no evidence of HACE1 mutations or deletions, but hypermethylation of two upstream CpG islands correlates with low HACE1 expression in tumor samples. Our findings implicate Hace1 as a novel ubiquitin-protein ligase and demonstrate that its expression is very low in primary Wilms' tumors.     

Antibody to type I insulinlike growth factor receptor inhibits growth of Wilms'' tumor in culture and in athymic mice.

The role of the type I insulinlike growth factor (IGF) receptor in regulating growth of Wilms' tumor (WT) was evaluated by examining the effect of antibody-mediated inhibition of this receptor on tumor growth in cell cultures and as heterotransplants in athymic mice. An antibody to the human type I IGF receptor (alpha IR-3) inhibited 125I-IGF-1 binding and prevented stimulation of thymidine incorporation by IGF-1 in vitro. Intraperitoneal administration of alpha IR-3 to nude mice bearing WT heterotransplants prevented tumor growth for 4 weeks and resulted in partial regression of established tumors. These data indicate the importance of IGF action in control of WT growth in vivo, and suggest potential therapeutic application using antigrowth factor receptor antibodies to block growth factor action.     

Unique insertional translocation in a childhood Wilms' tumor survivor detected when his daughter developed bilateral retinoblastoma

Retinoblastoma and Wilms' tumor are rare childhood embryonic tumors associated with loss or inactivation of tumor suppressor genes, RB1 located within 13q14, and WT1 located within 11p13. Interchromosomal insertional translocations occur rarely, and such rearrangements within RB1 or WT1, even rarer. We report a unique family in which an insertional translocation of a chromosomal segment that included band 13q14 inserted into 11p13 caused childhood Wilms' tumor in the father, and whose child developed bilateral retinoblastoma. This is the first case of an insertional translocation that caused both tumors. This insertional translocation had significant consequences for genetic counseling and in utero diagnosis. The estimated risk for an offspring of this father to develop Wilms' tumor is up to 50%, to develop retinoblastoma up to 25%, to have neither tumor 25%, and to have both tumors 0%.     

Responsiveness of chemotherapy based on the histological type and Wilms' tumor suppressor gene mutation in bilateral Wilms' tumor

To clarify a characteristic of bilateral Wilms' tumor (WT), we examined the clinical and histological features, chemotherapy response and mutations in Wilms' tumor suppressor gene (WT1) in five patients. Deoxyribonucleic acid was extracted from peripheral lymphocytes and tumor samples, and direct DNA sequencing was performed to detect WT1 mutations. Paraffin sections were stained with H&E for histological review and immunostained with anti-WT1, anti-Ki-67, anti-S-100 protein and antimyogenin antibodies. In contrast to the single case of epithelial-type WT, the other four cases were fetal rhabdomyomatous nephroblastoma (FRN) or contained a premature skeletal muscle component and appeared to be resistant to chemotherapy because there was no reduction in tumor volume. However, after chemotherapy, most of the tumor components changed into mature striated muscle cells, most of which immunostained almost completely negative for Ki-67. All four cases had the same point mutation of WT1. From our results, the histological findings correlated with WT1 mutations in bilateral WT. The tumor volume of FRN did not decrease in response to chemotherapy. It is possible to predict the chemotherapy response by examining bilateral WT for WT1 mutations and the histological characteristics of tumors.     

DNA免疫激发针对肾母细胞瘤CTL效应初步研究

目的 将鼠源肾母细胞瘤WT1基因一段序列(WT1y)构建于真核表达质粒pCDNA3.1( ),通过小鼠和体外细胞模型初步研究所激发的针对肾母细胞瘤CTL效应.方法 人工合成WT1基因一段序列,含有HLA-A*2402锚定残基的9个氨基酸.构建重组真核表达质粒pCDNA3.1( )/WT1y.免疫Balb/c小鼠,通过ELISA方法检测体液免疫反应;分离脾淋巴细胞,FCM检测脾淋巴细胞中CD4/CD8;将分离的脾淋巴细胞与小鼠肾母细胞瘤细胞共培养,检测其体外溶解组织相容性抗原型别一致的外源性靶细胞能力.结果 构建的重组质粒经测序鉴定,与GenBank中登录的序列完全一致;免疫组小鼠T淋巴细胞增殖及CTL活性均明显优于对照组(P＜0.01);体外具有较强溶解靶细胞的功能.结论 WT1基因的DNA疫苗初步研究具有很强的CTL效应,为小儿肾母细胞瘤的治疗提供了新的思路.     

The same mutation affecting the splicing of WT1 gene is present on Frasier syndrome patients with or without Wilms' tumor

Denys-Drash and Frasier syndromes are rare human disorders that associate nephropathy with gonadal and genital abnormalities. In DDS there is a predisposition to Wilms' tumor. Heterozygous point mutations in the Wilms' tumor, type1 gene (WT1), particularly those altering the zinc finger (ZF) encoding exons, have been reported in most DDS patients, while mutations in intron 9 of the same gene cause FS. This paper describes two cases of DDS, one FS and one patient with Wilm's tumor and intersex genitalia, in which mutations were searched by sequencing the exons 8 and 9 of WT1 gene. Patient 1 carried a missense point mutation in exon 8 (ZF2), converting a CGA-Arg codon to a TGA-stop codon. Patient 2 presented a single nucleotide deletion within exon 9 (ZF3) introducing a premature chain termination at codon 398. Patients 3 and 4 had a C-->T transition at position +4 of the second alternative splice donor site of exon 9 (this mutation was detected in peripheral blood and in tumor derived DNA of patient 3). However, patient 3 had previously developed a Wilms' tumor. This is the first case of Wilms' tumor development in a phenotypically and genetically confirmed case of FS.     

Cancer immunotherapy targeting Wilms' tumor gene WT1 product

The Wilms' tumor gene WT1 is expressed at high levels in leukemic blast cells in most acute myeloid and lymphoblastic leukemias. In myelodysplastic syndrome, WT1 mRNA expression levels increase along with disease progression; thus, WT1 mRNA is a tumor marker for leukemic blast cells. WT mRNA is also expressed at high levels in various types of solid cancers, including cancers of the lung, breast, colon and pancreas. Patients with WT1-expressing tumors produce antibodies and cytotoxic T-lymphocytes against WT1 protein, indicating that WT1 protein is highly immunogenic and a promising tumor antigen. Major histocompatibility complex class I-restricted cytotoxic T-lymphocyte and class II-restricted helper epitopes of WT1 protein were identified, and clinical studies of cancer immunotherapy using these cytotoxic T-lymphocyte epitope peptides were performed without significant adverse effect and with clinical results promising enough to encourage further clinical trials. The clinical efficacy of cancer immunotherapy targeting the WT1 protein should be clarified by a large-scale clinical study.     

Intra-abdominal desmoplastic small round cell tumor: immunohistochemical evidence for up-regulation of autocrine and paracrine growth factors

Desmoplastic small round cell tumors (DSRCT) are highly aggressive tumors typically involving the serosal surfaces of the peritoneum. Patients often present with abdominal pain, an abdominal mass, ascites or signs of intestinal obstruction. Cytogenetic and molecular studies have identified a characteristic t(11;22)(p13;q12) translocation within the tumor cells. The fused gene product apparently aligns the NH2-terminal domain (NTD) of the EWS gene to the zinc finger DNA-binding domain of the WT1 gene. This product could lead to loss of the tumor suppressor effect of the WT1 gene as well as to an increase in EWS driven expression of growth factors normally repressed by WT1. We investigated this latter possibility by performing immunohistochemical studies on formalin fixed tissue from 10 cases of DSRCT and five Wilms' tumors using antibodies to insulin-like growth factor (IGF)-II, the latency associated peptide of transforming growth factor (TGF)-beta1, platelet-derived growth factor (PDGF)-AB chain and PDGF-alpha receptor, respectively. In general, tumor cells were strongly positive for these growth factors in DSRCT, while stromal cells were negative for IGF-II and positive for the other growth factors in parallel with the tumor cells. Wilms' tumor cells were essentially negative for PDGF-AB chains, but positive for IGF-II, and the latency associated peptide of TGF-beta1 and variably positive for PDGF-alpha receptor. These findings support the proposed molecular mechanism of tumorigenesis for DSRCT and may help explain this tumor's poor prognosis.     

Presence of the long chain form of polysialic acid of the neural cell adhesion molecule in Wilms'' tumor. Identification of a cell adhesion molecule as an oncodevelopmental antigen and implications for tumor histogenesis.

The long chain form of polysialic acid characteristic of the low adhesive embryonic form of the neural cell adhesion molecule NCAM is temporally and spatially expressed in developing kidney but undetectable in normal adult kidney. Therefore, this molecule represents a developmentally regulated antigen in kidney contrasted with neural tissue, where it is also detectable in the adult brain. This investigation of 25 Wilms' tumors comprising all different histologic types demonstrates expression of this molecule under conditions of malignant growth. Immunostaining was observed in Wilms' tumors with both a monoclonal anti-polysialic acid antibody and a polyclonal anti-NCAM polypeptide antiserum. Intense cell surface staining sensitive to endosialidases specifically hydrolyzing alpha 2,8 linked (poly)sialic acid was detectable in blastemal regions, and weaker, variable labeling was seen over tubules and glomeruloid bodies. The stroma was not stained. This is evidence indicating that Wilms' tumor originates from the embryonic equivalent of induced metanephrogenic mesenchyme. It seems unlikely however, that the stroma is derived from the blastema. The same high molecular mass broad band typical of the embryonic form of NCAM was revealed by immunoblot analysis of homogenates from Wilms' tumor as well as from embryonic kidney and brain. In situ hybridization demonstrated the presence of mRNA for NCAM in all but stromal elements of Wilm's tumors. Thus, polysialic acid is present on NCAM and represents a new oncodevelopmental antigen in human kidney. Polysialic acid was greatly reduced or absent by immunohistochemistry and immunoblotting in necrotic tumor areas.     

Expression of the Wilms'' tumour gene WT1 in the developing human and in paediatric renal tumours: an immunohistochemical study.

AIMS: The Wilms' tumour gene (WT1) product is expressed during the development of the urogenital system. This study was undertaken to evaluate four anti-WT1 antibodies and use the most specific one to examine the expression of WT1 in formalin fixed, paraffin wax embedded tissues from human embryos, fetuses, and paediatric renal neoplasms. METHODS: The antibodies were assessed on paraffin sections of fetal kidney and by western blotting. Immunohistochemical techniques were optimised and performed on a range of embryonic, fetal, and infant tissues from 35 days post-conception to three months of age, and on a selection of paediatric renal neoplasms. RESULTS: The antibodies tested were found to vary in their specificity. Anomalous expression in smooth muscle was seen with one batch of a commercial polyclonal antibody. WT1 protein was detected in both the metanephros and the mesonephros, the spleen, the gonads, and in the peritoneal mesothelium in fetuses. WT1 was expressed in nuclei and was strongest in the podocytes of fetal kidney. The podocytes of infant glomeruli were also positive. There was focal positive staining in Wilms' tumours, nephrogenic rests, and in a cystic partially differentiated nephroblastoma. Staining of nuclei was seen in one of two rhabdoid tumours of the kidney. No positive staining was seen in other renal tumours. CONCLUSIONS: WT1 is detected readily in formalin fixed material. There were differences in specificity between batches of the polyclonal antibodies used. The distribution of the WT1 gene product in tissues and tumours reflected previous findings with in situ hybridisation studies of WT1 mRNA.     

Genomic changes in the WT-gene (WT1) in Wilms' tumors and their correlation with histology.

The authors studied genomic changes in unilateral Wilms' tumors by using WT33, a candidate cDNA for the tumor, and their correlation with histology. By Southern blot analysis, three cases of genomic deletions of both alleles were found in 25 tumors. The three tumors that showed genomic deletions were histologically classified as triphasic nephroblastic Wilms' tumor and one of them was associated with intralobar nephroblastomatosis and a rhabdomyomatous component. In one case, the WT1 gene was totally deleted, in another case, the 3' region of the gene was partially deleted, and in the last one, the deletion of DNA was intragenic. This is the first report of a comparison of genomic alteration with histopathology. These findings show new aspects of the role of the WT1 gene in the development of Wilms' tumor.