Primary renal neoplasms with the ASPL-TFE3 gene fusion of alveolar soft part sarcoma: a distinctive tumor entity previously included among renal cell carcinomas of children and adolescents.

The unbalanced translocation, der(17)t(X;17)(p11.2;q25), is characteristic of alveolar soft part sarcoma (ASPS). We have recently shown that this translocation fuses the TFE3 transcription factor gene at Xp11.2 to ASPL, a novel gene at 17q25. We describe herein eight morphologically distinctive renal tumors occurring in young people that bear the identical ASPL-TFE3 fusion transcript as ASPS, with the distinction that the t(X;17) translocation is cytogenetically balanced in these renal tumors. A relationship between these renal tumors and ASPS was initially suggested by the cytogenetic finding of a balanced t(X;17)(p11.2;q25) in two of the cases, and the ASPL-TFE3 fusion transcripts were then confirmed by reverse transcriptase-polymerase chain reaction. The morphology of these eight ASPL-TFE3 fusion-positive renal tumors, although overlapping in some aspects that of classic ASPS, more closely resembles renal cell carcinoma (RCC), which was the a priori diagnosis in all cases. These tumors demonstrate nested and pseudopapillary patterns of growth, psammomatous calcifications, and epithelioid cells with abundant clear cytoplasm and well-defined cell borders. By immunohistochemistry, four tumors were negative for all epithelial markers tested, whereas four were focally positive for cytokeratin and two were reactive for epithelial membrane antigen (EMA) (one diffusely, one focally). Electron microscopy of six tumors demonstrated a combination of ASPS-like features (dense granules in four cases, rhomboid crystals in two cases) and epithelial features (cell junctions in six cases, microvilli and true glandular lumens in three cases). Overall, although seven of eight tumors demonstrated at least focal epithelial features by electron microscopy or immunohistochemistry, the degree and extent of epithelial differentiation was notably less than expected for typical RCC. We confirmed the balanced nature of the t(X;17) translocation by fluorescence in situ hybridization in all seven renal tumors thus analyzed, which contrasts sharply with the unbalanced nature of the translocation in ASPS. In summary, a subset of tumors previously considered to be RCC in young people are in fact genetically related to ASPS, although their distinctive morphological and genetic features justify their classification as a distinctive neoplastic entity. Finally, the finding of distinctive tumors being associated with balanced and unbalanced forms of the same translocation is to our knowledge, unprecedented.

Advances in cytochemical methods for detection of apoptosis.

In an earlier article from this laboratory, the current methods developed to detect apoptosis in cells and tissues were highlighted, along with the challenges in their interpretation. Recent discoveries concerning the underlying biochemical mechanisms of apoptotic effector pathways have made possible further assays that allow a more direct measure of the activation of the apoptotic machinery in cells. This article summarizes some of these newer methods and extends the interpretation of the more classical assays of apoptosis in a defined cell system. We present data in KB and PC3 cell model culture systems induced to undergo apoptosis by the plant toxin ricin. Using a modified in situ nick translation assay (ISNT) with either Bodipy or BUdR labeling, we confirm that most cells showing altered nuclear morphology do not show reactivity with this assay until very late in the apoptotic process. We also show that only a minority of cells label with fluorescent annexin V during apoptosis but that apoptotic cells continue to internalize material from the cell surface through endocytosis after becoming reactive with annexin V. In addition, we describe the utility of a prototype of new assays for caspase substrate cleavage products, the detection of cleaved cytokeratin 18. It is these newer cleavage product assays that perhaps hold the greatest promise for specific detection of apoptosis in cells either in cell culture or in intact tissues. (J Histochem Cytochem 49:821-832, 2001)

Prognostic significance of Bcl-2 in Wilms' tumor and oncogenic potential of Bcl-X(L) in rare tumor cases.

Anaplastic Wilms' tumors are commonly believed to be rare forms of progression, driven by p53 mutations, of the more common classical Wilms' tumor or nephroblastoma Contrary to classical Wilms' tumors, anaplastic tumors traditionally tend to metastasize, to be drug-resistant and to have a poor prognosis. The Bcl-2 gene product protects cells from programmed cell death, and its over-expression has been proposed to be tumorigenic and to mediate resistance to therapy. Because Bcl-2 has been reported to be transcriptionally repressed by p53, using immuno-histochemistry and mRNA analyses, we have examined Bcl-2 expression in a panel of 10 classical and 3 anaplastic nephroblastomas in which the p53 status had been previously analyzed. We found that classical Wilms' tumors expressed significant amounts of Bcl-2 mRNA and protein, whereas anaplastic tumors did not, regardless of p53 mutations. However, because mortality occurred both among patients with classical and among those with anaplastic tumors, which had divergent Bcl-2 expression, analysis of variance failed to demonstrate prognostic Bcl-2 significance. Therefore, we examined the expression of the Bcl-X and Bax genes, which are known to synergize and antagonize Bcl-2, respectively. With the exception of anaplastic tumor W17, the monotony of Bcl-X and Bax mRNA levels did not suggest that the expression of these apoptosis-regulating genes could have a role in the prognosis of nephroblastoma. In addition to the standard 2.7-kb Bcl-X(L) mRNA, W17 expressed a 3.5-kb mRNA species which had the same coding capacity for Bcl-X(L) as the 2.7-kb mRNA. Western analysis demonstrated that W17 had the highest level of Bcl-X(L) protein, suggesting that Bcl-X(L) over-expression could play a part in the development of anaplasia in rare Wilms' tumor cases without affecting prognosis.

Inhibition of cellular proliferation by the Wilms tumor suppressor WT1 requires association with the inducible chaperone Hsp70.

The Wilms tumor suppressor WT1 encodes a zinc finger transcription factor that is expressed in glomerular podocytes during a narrow window in kidney development. By immunoprecipitation and protein microsequencing analysis, we have identified a major cellular protein associated with endogenous WT1 to be the inducible chaperone Hsp70. WT1 and Hsp70 are physically associated in embryonic rat kidney cells, in primary Wilms tumor specimens and in cultured cells with inducible expression of WT1. Colocalization of WT1 and Hsp70 is evident within podocytes of the developing kidney, and Hsp70 is recruited to the characteristic subnuclear clusters that contain WT1. The amino-terminal transactivation domain of WT1 is required for binding to Hsp70, and expression of that domain itself is sufficient to induce expression of Hsp70 through the heat shock element (HSE). Substitution of a heterologous Hsp70-binding domain derived from human DNAJ is sufficient to restore the functional properties of a WT1 protein with an amino-terminal deletion, an effect that is abrogated by a point mutation in DNAJ that reduces binding to Hsp70. These observations indicate that Hsp70 is an important cofactor for the function of WT1, and suggest a potential role for this chaperone during kidney differentiation.

Establishment and molecular characterization of five cell lines derived from renal and extrarenal malignant rhabdoid tumors.

Malignant rhabdoid tumor (MRT) is a rare, enigmatic childhood cancer characterized by extreme aggressiveness and resistance to chemotherapy. To understand better the origin of the tumor and the mechanisms by which it develops and resists treatment, five cell lines were established from patients presenting with MRT (two renal and three extrarenal tumors). All of the cell lines display the light microscopic and ultrastructural features, as well as the variable immunohistochemical profile, characteristic of MRT. All are capable of forming tumors in nude mice. Three of the cell lines have detectable abnormalities of chromosome 22: one a t(22, 22) unbalanced translocation and two others a loss of heterozygosity of polymerase chain reaction-based microsatellite markers. Northern blot analysis showed that overexpression of the c-myc message was a consistent characteristic of the five MRTs evaluated. Although mutations of the p53 gene were not detectable by sequence analysis, all of the cell lines showed nuclear accumulation of the p53 protein by an immunocytochemical analysis in a minority of the cells. This result suggests that dysfunction in a p53-dependent apoptotic pathway might play a role in the multiple drug resistance phenotype of these tumors.

Induction of p21 by the Wilms' tumor suppressor gene WT1.

WT1 encodes a zinc finger transcription factor that is expressed in the developing kidney and the inactivation of which leads to Wilms' tumor, a pediatric kidney cancer. We have recently shown that inducible expression of WT1 in osteosarcoma cells triggers programmed cell death, an effect that is associated with transcriptional repression of the endogenous epidermal growth factor receptor. We now show that WT1-mediated apoptosis is preceded by induction of the cyclin-dependent kinase inhibitor p21, associated with G1 phase arrest. This effect is only demonstrated by WT1 isoforms with an intact DNA binding domain, and it is associated with increased expression of endogenous p21 mRNA. WT1-mediated induction of p21 is independent of p53, another tumor suppressor gene known to regulate p21 expression. In the kidney, p21 is expressed in differentiating glomerular podocytes along with WT1. We conclude that induction of p21 expression may contribute to WT1-dependent differentiation pathways in the kidney and potentially to the function of WT1 as a tumor suppressor gene.

Anaplasia and drug selection-independent overexpression of the multidrug resistance gene, MDR1, in Wilms' tumor.

One reason for the failure of chemotherapy is the overexpression of the multidrug resistance gene, MDR1. The product of this gene is the multidrug transporter P-glycoprotein, an ATP-dependent pump that extrudes drugs from the cytoplasm. Some tumors inherently express P-glycoprotein, whereas others acquire the ability to do so after exposure to certain chemotherapeutic agents, often by the mechanism of gene amplification. Classical Wilms' tumors (nephroblastoma) typically respond to therapy and have a good prognosis. On the contrary, anaplastic Wilms' tumors are generally refractory to chemotherapy. These anaplastic variants are rare (4.5% of all Wilms' tumors reported in the United States), aggressive, and often fatal forms of tumor, which are commonly thought to result from the progression of classical Wilms' tumors. To investigate the basis for this differential response to therapy, we examined a number of classical and anaplastic Wilms' tumors for the expression of the MDR1 gene by immunohistochemical and mRNA analysis. Classical Wilms' tumors consistently did not express P-glycoprotein except in areas of tubular differentiation, as in normal kidney. Similarly, two of three anaplastic tumors failed to show P-glycoprotein expression. In contrast, cultured cells derived from a third anaplastic tumor, W4, exhibited strong P-glycoprotein expression and were drug resistant in vitro. Southern analysis revealed that W4 cells contained a single copy of the MDR1 gene per haploid genome similar to normal cells, demonstrating that the overexpression of MDR1 was not caused by gene amplification. Transcriptional activation of the MDR1 gene would be in keeping with the concept that p53 might act as a transcriptional repressor of the MDR1 gene.

A comparative study of apoptosis and cell proliferation in infantile and adult fibrosarcomas.

The infantile fibrosarcoma, a rare tumor phenotypically similar to the adult fibrosarcoma, frequently has a benign course marked by spontaneous regression. Because biologic mechanisms responsible for this regression remain unexplained, an investigation of the role of apoptotic cell death is warranted. The rate of apoptotic cell death has been compared in five cases each of infantile and adult fibrosarcoma by quantitative estimation of in situ DNA double strand breaks. Although positively stained apoptotic cells are evident in all 10 cases, the apoptotic index is significantly higher in infantile cases (mean 6.6% +/- 0.80) compared to adult cases (mean 0.5% +/- 0.08). The proliferative (MIB-1) index of each specimen has been calculated by immunostaining for cell cycle phase-dependent Ki-67 antigen with MIB-1 antibody. Infantile cases have a significantly lower proliferative (MIB-1) index (mean 0.4 +/- 0.15) than adult counterparts (mean 15.9 +/- 3.76). The relatively benign course of the infantile fibrosarcoma may be due to two factors--a significantly lower proliferative (MIB-1) index coupled with enhanced apoptosis.

mhatomic pathology turnaround times. Use and abuse.

Anatomic pathology service is judged by accuracy of diagnosis and rapidity of reporting of that diagnosis to clinicians. Turnaround times have been used in anatomic pathology to show how long it take to produce a tissue report. Rapid tissue diagnosis is important to patients, clinicians, administrators, and anatomic pathologists. Documentation of turnaround times (TAT) to substantiate efficient service can be similarly important. The authors describe how turnaround times can be used and discuss their impact from different perspectives.

All-trans-retinoic acid-induced growth suppression of blastemal Wilms' tumor.

All-trans-retinoic acid (RA) has been used to suppress growth of malignant cells and induce epithelial differentiation. We investigated whether RA had a similar effect on Wilms' tumor, a childhood tumor of the kidney that arises from the undifferentiated metanephric blastema. W13 cells, a cell line derived from a blastemal Wilms' tumor, were exposed to RA (10(-9)-10(-5) M) and its effects on cell proliferation, gene expression, and differentiation were examined. Treatment of W13 cells with RA resulted in a dose-dependent suppression of growth. Changes in expression of selected genes were determined by Northern analysis. After 24 h, there was a marked dose-dependent down-regulation of N-myc mRNA as well as up-regulation of insulin-like growth factor-II (IGF-II) mRNA. [125I]IGF-II ligand blotting of conditioned medium from RA-treated cultures revealed a dramatic alteration in the pattern of expression of insulin-like growth factor binding proteins (IGFBPs). Examination of RA-treated W13 cultures by light and electron microscopy did not reveal appreciable morphological changes. We conclude that RA inhibits growth and alters gene expression of W13 cells without inducing epithelial differentiation. The modulation of expression of IGF-II, IGFBP, and N-myc may play a role in RA-induced growth suppression of Wilms' tumor cells.

Molecular biology and the pediatric surgeon: definitions and basic methodology.

Molecular biology techniques and their application are becoming increasingly important to the practicing clinician. This article reviews the basics of DNA chemistry and highlights important molecular biology techniques. It will provide a guide for the pediatric surgeon as she/he attempts to integrate this field into everyday practice.

Inhibition of insulin like growth factor II autocrine growth of Wilms' tumor by suramin in vitro and in vivo.

Suramin was found to affect the Wilms' tumor (WT) cell line, W13, by inhibiting in vitro growth (half-maximal inhibitory dose (ID50)=11 microM), insulin like growth factor II (IGF-II) cell binding (ID50 = 10 microM) and IGF-II induced DNA synthesis (ID50 = 8 microM). In addition, suramin inhibited cross-linking of [125I]IGF-II to the type 1 IGF receptor (IGF1R) and type 2 IGF receptor (IGF2R). Disruption of IGF-II/IGF1R interaction appears to be the main mode of action of suramin since the suramin response was abolished in the presence of the IGF1R blocking antibody, alpha IR-3. When administered to athymic mice bearing W13 heterotransplants, suramin suppressed the linear tumor growth rate by 64%.

Immunophenotype, mRNA expression, and gene structure of p53 in Wilms' tumors.

The anaplastic variant of Wilms' tumor is regarded as the result of tumor progression of the more common classic Wilms' tumor. Anaplasia is rare and occurs in only 4.5% of tumors. Three anaplastic Wilms' tumors in our collection were examined in comparison with 10 classic Wilms' tumors for p53 expression by immunohistochemical techniques and Northern blot analysis, and their p53 gene structure was determined by single-stranded conformation polymorphism and sequence analysis. All classic tumors contained a wild-type p53 gene and expressed marginal levels of protein as expected for normal p53. In contrast, three out of three anaplastic tumors demonstrated evidence of p53 alterations consistent with a role of p53 in tumor progression. One of the anaplastic mutants (W4) did not express protein or p53 mRNA. Its apparently normal immunophenotype would have disguised the mutated nature of p53, which was detected only by mRNA and sequence analysis. The second anaplastic mutant (W16) contained normal levels of p53 mRNA, but overexpressed the protein in a fashion typical of mutated p53. The same immunophenotype was displayed by fixed primary tissue of the third anaplastic tumor (W17), but p53 mutation could not be confirmed for lack of frozen primary material. The present study emphasizes the importance of p53 function in the anaplastic progression of Wilms' tumor and the risk of error in assessing normal p53 function using a single methodology.

WT1 induces expression of insulin-like growth factor 2 in Wilms' tumor cells.

The Wilms' tumor suppressor gene WT1 encodes a zinc finger transcription factor, whose expression inhibits the growth of the RM1 Wilms' tumor cell line. Transient transfection of WT1 constructs into 3T3 or 293 cells results in transcriptional repression of a number of cotransfected promoters containing the early growth response gene 1 consensus sequence. We now show that WT1 has properties of a transcriptional activator in RM1 cells, an effect that may be associated with the presence of a mutated p53 gene in these cells. Stable transfection of wild-type WT1 into RM1 cells results in induction of endogenous insulin-like growth factor 2 (IGF2) but not of other previously postulated WT1-target genes. The induction of IGF2 is dramatically enhanced by WT1 mutants encoding an altered transactivation domain. We conclude that IGF2 is a potentially physiological target gene for WT1 and that its induction may contribute to the growth-stimulating effects of WT1 variants.

WT1 suppresses synthesis of the epidermal growth factor receptor and induces apoptosis.

The Wilms tumor suppressor gene WT1 encodes a developmentally regulated transcription factor that is mutated in a subset of embryonal tumors. To test its functional properties, we developed osteosarcoma cell lines expressing WT1 under an inducible tetracycline-regulated promoter. Induction of WT1 resulted in programmed cell death. This effect, which was differentially mediated by the alternative splicing variants of WT1, was independent of p53. WT1-mediated apoptosis was associated with reduced synthesis of the epidermal growth factor receptor (EGFR), but not of other postulated WT1-target genes, and it was abrogated by constitutive expression of EGFR. WT1 repressed transcription from the EGFR promoter, binding to two TC-rich repeat sequences. In the developing kidney, EGFR expression in renal precursor cells declined with the onset of WT1 expression. Repression of EGFR and induction of apoptosis by mechanism that may contribute to its critical role in normal kidney development and to the immortalization of tumor cells with inactivated WT1 alleles.

Characterization of a monoclonal antibody recognizing the blastemal element of Wilms' tumors and fetal kidneys.

A blastema-associated antigen (BLA-1) was detected using a monoclonal antibody against malignant blastema from a Wilms' tumor. The localization of BLA-1 was investigated in a series of nine Wilms' cases, five fetal, one childhood, and two adult kidneys. In this series, BLA-1 antibody consistently stained cell surfaces of all Wilms' tumors containing blastemal components. The same staining pattern was maintained in tumors grown as heterotransplants in nude mice. The expression of BLA-1 antigen was examined in normal blastema of fetal kidneys. BLA-1 was immunolocalized to condensed blastemal cells in the nephrogenic zone throughout gestation. In addition, kidney samples from a young child or adults contained no blastemal cells and therefore showed no blastemal cell surface staining. Glomerular mesangial cell staining was demonstrated in kidneys from 12 weeks of gestation through adulthood. This staining in developing and mature glomeruli implies that mesangial cells may be derived from condensed blastemal cells. The finding of a cell surface antigen common to Wilms' blastema, fetal blastema, and mesangial cells has not been previously demonstrated.

Characterization of a monoclonal antibody recognizing selective epithelial elements of Wilms' tumors and fetal kidneys.

A new antigen was detected using a monoclonal antibody generated against malignant blastema from a Wilms' tumor. This antigen showed variable expression in malignant blastemal cells but was never detected in normal blastema of fetal kidneys irrespective of gestational stage. In a series of 16 Wilms' tumors, the most intense and consistent staining was seen in tubule-associated epithelial cells. Such tubular staining is not surprising as the putative induction of malignant blastema to differentiate into malignant tubules is thought to parallel normal tubulogenesis. This antigen was also associated with epithelial cells located in a variety of fetal kidney structures. Again, the staining was most consistent in tubular epithelia. This monoclonal antibody reactive with a blastemal-epithelial-tubular (BET) antigen should be of value in studying the induction of epithelial differentiation in the normal and diseased human kidney.

Expression of insulin-like growth factor binding protein 2 (IGFBP-2) in Wilms' tumors.

Human Wilms' tumor (WT) expresses insulin-like growth factor (IGF) II and its cognate receptor, type 1 IGF receptor, forming a self-stimulating "autocrine loop." The biological activity of IGF-II is modulated by a class of soluble receptors called IGF binding proteins (IGFBP). To determine if IGFBP play a role in the biology of WT, extracts of nude mouse heterotransplants of three blastemal WT were examined for the ability to bind radiolabeled IGF-II by ligand blot analysis. [125I]IGF-II bound to a protein of M(r) 35 kDa. To confirm that this binding protein was being expressed by the tumor itself and not background from the host, tumor explants were prepared in cell culture. Conditioned culture media from blastemal WT cell cultures were found to contain the 35-kDa IGFBP. This secreted binding protein was identified as IGFBP-2 by screening for reactivity to characterized IGFBP antisera. Total RNA from primary WT or WT cells in culture was examined for expression of IGFBP-2 mRNA using an RNase protection assay. All three WT expressed IGFBP-2 mRNA. These data suggest a role for IGFBP-2 in the IGF-II-dependent growth of Wilms' tumor and in the developing kidney.

Serum-free culture and characterization of renal epithelial cells isolated from human fetal kidneys of varying gestational age.

Cell cultures were initiated from seven human fetal kidneys that varied in gestational age from 90 days to newborn. The growth medium utilized was a 1:1 mixture of Dulbecco's modified Eagle's and Ham's F12 supplemented with selenium (5 ng/ml), insulin (5 micrograms/ml), transferrin (5 micrograms/ml), hydrocortisone (36 ng/ml), triiodothyronine (4 pg/ml), and epidermal growth factor (10 ng/ml). For all the kidney isolates, initial cell attachment occurred within 12 h through multicell spheroids, and by 24 h a rapidly growing population of cells was obtained. Confluency was reached within 3 to 6 days. A combination of light microscopy, immunohistochemistry, and ultrastructural evaluation was utilized to characterize the resulting cultures as epithelial and homogeneous within each isolate and among the isolates. That is, regardless of gestational age of the fetal kidney used as starting material, an identical or highly similar population of cells was obtained. By light microscopy, the cultures were noted to form very few domes, the number being an indication of transport activity. However, ultrastructural examination revealed that the cells were noted to form domes composed of only a few cells or "micro-domes" that would not be visible by light microscopy. Within the micro-domes as well as other areas of the monolayer an apparent absence of tight junctions was noted by routine transmission electron microscopy. However, by freeze fracture analysis cells were shown to possess sealing strands, the structural component of tight junctions. It is postulated that the tight junctions of fetal epithelial cells are structurally altered as compared to tight junctions in adult renal epithelial cell cultures.