Inactivation of WT1 in nephrogenic rests, genetic precursors to Wilms' tumour.

Nephrogenic rests consist of foci of primitive renal cells, typically microscopic, that are found within the normal kidney tissue of children with Wilms' tumour. To study the relationship between nephrogenic rests and the associated tumours, we screened these lesions for mutations in the 11p13 Wilms' tumour suppressor gene, WT1. In two cases in which the Wilms' tumour contained a somatic WT1 mutation, the nephrogenic rest had the identical mutation. Nephrogenic rests and Wilms' tumours are therefore topographically distinct lesions that are clonally derived from an early renal stem cell. Inactivation of WT1 appears to be an early genetic event which can lead to the formation of nephrogenic rests, enhancing the probability that additional genetic hits will lead to Wilms' tumour.

Immunohistochemical demonstration of IgG in Reed-Sternberg and other cells in Hodgkin's disease.

Spleens and lymph nodes fixed lightly for optimal immunocytochemistry or processed routinely for surgical diagnosis disclosed strong selective immunostaining for IgG in numerous immunocytes in tumor-free areas. Areas involved by Hodgkin's disease revealed, in addition, strong immunostaining for IgG but not IgM in Reed-Sternberg cells and faint to strong staining in Hodgkin cells as well. Ultrastructurally the Reed-Sternberg and Hodgkin cells displayed abundant polyribosomes and sparse granular reticulum and appeared to form unexportable IgG on unbound ribosomes.

WT1-mediated growth suppression of Wilms tumor cells expressing a WT1 splicing variant.

A human Wilms tumor cell line (RM1) was developed to test the tumor suppressor activity of WT1, a zinc finger transcription factor that is expressed in the developing human kidney and is mutationally inactivated in a subset of Wilms tumors. Transfection of each of four wild-type WT1 isoforms suppressed the growth of RM1 cells. The endogenous WT1 transcript in these cells was devoid of exon 2 sequences, a splicing alteration that was also detected in varying amounts in all Wilms tumors tested but not in normal kidney. Production of this abnormal transcript, which encodes a functionally altered protein, may represent a distinct mechanism for inactivating WT1 in Wilms tumors.

In vitro culture of cells exfoliated in the urine by patients with diabetes mellitus.

As an approach to facilitate the understanding of the progression of diabetic renal disease, we assessed the urine of diabetic patients and normal volunteers for the presence of cells that could be cultured in vitro. The results suggest that both normal control subjects and diabetic patients, without clinically detectable microangiopathy, exfoliate few culturable cells into the urine. In contrast, diabetics with documented retinopathy but without nephropathy exfoliate substantially higher numbers of culturable cells (5.2 cells/100 ml urine), whereas diabetics with both retinopathy and advanced nephropathy exfoliate even greater numbers of culturable cells (50.8 cells/100 ml urine). The cells that are exfoliated and culturable can be divided into five distinct cell types based on morphology at the light microscope level. The exfoliated cells proliferate at clonal density after isolation from urine and are epithelial in appearance. These data suggest that the culture of cells from urine might have diagnostic value as an early indicator of diabetic renal disease and provide a convenient, noninvasive new source of human kidney epithelial cells.

Multilaminar endoplasmic reticulum and abnormal mitosis in Hodgkin tumor cells.

A multilaminar alteration of endoplasmic reticulum (ER) has been observed in tumor cells of eight patients with Hodgkin's disease and a patient with histiocytic lymphoma. These multilaminar structures are more numerous in dividing cells and thus appear to arise primarily during mitosis. The stacked membranes in the multilaminar structures possibly result from abnormal sticking of organelle membranes, as evidenced in this study of adherence of ER to other elements of ER, nuclear envelope, mitochondria, or lipid droplets. Multilaminar ER was identified in all mitotic tumor cells, a rare mitotic plasma cell, and numerous interphase Hodgkin cells. The paucity of multilaminar ER in normal mitotic cells and its virtual absence for normal interphase cells suggest that this structure represents a pathological alteration in tumor cells from patients with Hodgkin's disease and histiocytic lymphoma. The multilaminar defect of ER is associated with other atypical features of ER in Hodgkin tumor cells, including the excessive length and curving of ER profiles, the collapse of the ER cisternae, and the overall sparsity of this organelle. Other abnormalities observed in mitotic Hodgkin tumor cells include the presence of disorganized microtubules, large cytoplasmic vacuoles, and abnormally clumped chromosomal material and the persistence throughout mitosis of bodies suggestive of nucleoli and of the nuclear bodies of interphase cells.

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.

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.

The Rappaport classification of non-Hodgkin's lymphomas: a closer look using other proposed classifications.

An essential purpose of a pathologic classification of non-Hodgkin's lymphomas is to supply guidance in the clinical management of patients. Ideally, an optimal subclassification should also be scientifically accurate, highly reproducible, and readily teachable. Such a system, when used in conjunction with uniform staging, should enable relatively homogeneous groups of patients to be defined. In the present study, we have evaluated four new systems for possible additions to the traditional Rappaport classifcation. In response to specific questions the following tentative conclusions could be drawn: (1) Within the Rappaport nodular lymphomas, there are no differences in survival between lymphomas that are totally nodular verusus those that are nodular and diffuse. (2) In lymphomas composed of small cleaved follicular center cells of the Lukes-Collins system, survival appears to be independent of pattern (follicular, follicular and diffuse, or diffuse). In contrast, in tumors classified as centroblastic-centrocytic in the Kiel classification or those classified as large cleaved or large noncleaved in the Lukes-Collins system, a totally or partially follicular pattern confers a better prognosis than its diffuse counterpart. (3) The numbers are small but there is no apparent difference in survival between cases of Rappaport's difuse well differentiated lymphocytic lymphomas with or without plasmacytoid differentiation. (4) Within the original Rappaport DPDL there were at least two distinct types of lymphomas: (1) a convoluted lymphoblastic that occurs in younger patients has a high frequency of B symptoms and carries a poor prognosis; and (2) a diffuse lymphoma that is cytologically identical to nodular PDL, occurs in older patients, and has a relatively good prognosis. In August of 1976 Rappaport modified his classification to recognize these lymphoblastic lymphomas as a distinct clinicopathologic entity. (5) In this 22-yr retrospective review, neither the Kiel nor the Lukes-Collins system could identify any relatively favorable subsets within Rappaport's category of diffuse histiocytic lymphoma. Prospective studies applying the same approach to large numbers of patients subjected to modern uniform staging and aggressive combination chemotherapy may provide data upon which to base an optimal subclassification of DHL.

The occurrence of a peripheral T-cell lymphoma in a chronically immunosuppressed renal transplant patient.

A 32-year-old man received a cadavaric renal transplant in 1975 for end-stage renal disease and, thereafter, was treated with azathioprine and methylprednisolone for chronic immunosuppression. In 1985, he presented with fever and pancytopenia that persisted despite withdrawal of the immunosuppressive agents. Lymph node and liver biopsies demonstrated malignant lymphoma within the sinuses of the node and the sinusoids of the liver. A splenectomy was performed for persistent pancytopenia, and the spleen demonstrated malignant lymphoma of the diffuse mixed large and small cell type exclusively within the cords of the red pulp. The immunophenotype of the tumor cells was obtained by frozen section immunoperoxidase staining with monoclonal antibodies and flow cytometric analysis. The tumor cells were positive for the Pan T cell markers CD3 and CD2, but were negative for the subset markers CD4 and CD8. A DNA hybridization study conducted on the splenic tissue conclusively identified the clonal nature of the malignant T cells by demonstrating rearrangement of the T cell receptor beta gene. In spite of multiple chemotherapeutic regimens, the patient developed increasing peripheral blood involvement and died with disseminated lymphoma. This case appears to be unique in that it is the first report of a chronically immunosuppressed transplant recipient to develop a malignant lymphoma of the mature T cell type, and several of the pathologic features of the tumor have not been observed previously.

Aggressive bladder carcinoma in an adolescent. Report of a case with immunohistochemical, cytogenetic, and flow cytometric characterization.

Malignant bladder neoplasms of urothelial origin are rare among children; fewer than 125 cases have been reported. Typically, these tumors are single papillary lesions of low grade and stage that have an excellent prognosis following surgical excision. A grade III transitional cell carcinoma of the bladder occurred in a 14-year-old boy who had no urinary tract malformation, carcinogenic exposure, or family history of cancer. Immunohistochemical stains of the tumor were positive for cytokeratin and high-molecular-weight keratin. The tumor tissue failed to stain with an antibody to the patient's blood group [anti-ABO(H)] but was positive for the Thomsen-Frieden-reich antigen. Flow cytometry of the tumor cells demonstrated a diploid or near-diploid DNA content. A karyo-type of the tumor showed a modal chromosome number of 46 with one reciprocal translocation between chromosomes 17 and 22 and a nonreciprocal translocation between chromosomes 18 and 22. The tumor was unique because of its highly aggressive nature and its diploid chromosome number. This case represents the first indepth characterization of a transitional cell carcinoma in a pediatric patient by flow cytometry and cytogenetics, as well as a variety of immunohistochemical studies including ABO(H) blood group and Thomsen-Freidenreich antigens.

Immune complex receptors on cell surface. I. Ultrastructural demonstration of macrophages.

A method is described for ultrastructural localization of immune complex receptors on the surface of viable peritoneal exudate cells. The technique entails incubation with a soluble complex of horseradish peroxidase (HRP) and specific antibody to HRP at 4 degrees C followed by exposure to diaminobenzidine and processing for electron microscopy. The bound immune complexes were evident as focal deposits of HRP reaction product, adhering closely to the external surface of macrophages with an uninterrupted periodicity varying between 30 and 120 nm. Following incubation with an insoluble immune complex containing a higher proportion of antibody, receptor sites stained frequently, but large aggregates adhered to the cells. Rinsing cells after staining with soluble complexes partially displaced the bound immune complexes. Fixation prior to exposure to immune complexes largely eliminated the binding capacity of the immune complex receptors.

Cytochemical differentiation of nucleic acids with a Schiff-methylene blue sequence.

A method is described whereby a Feulgen type of hydrolysis of deoxyribonucleic acid is carried out on paraffin sections of routinely fixed tissues by controlled exposure of the sections to Bouin's fluid. Subsequent staining with Schiff reagent followed by methylene blue distinguishes red-to purple-stained deoxyribonucleic acid from blue-stained ribonucleic acid. This Schiff-methylene blue sequence visualizes ribonucleic acid in nucleoli and the chromidial substance of various normal and neoplastic cells and provides an assessment of their protein synthetic activity. The method has proved valuable in demonstrating normal immunocytes and immunoglobulin-forming tumor cells in pathologic specimens.

Immune complex receptors on cell surfaces. III. Topography of macrophage receptors demonstrated by new scanning electron microscopic peroxidase marker.

Receptors for immune complexes have been localized on rabbit alveolar macrophages with scanning electron microscopy by exposing the cells first to a soluble immune complex composed of horseradish peroxidase and antibody to horseradish peroxidase, and then incubating with a benzidine-containing substrate that yields crystalline reaction product. Receptors were visualized by this means as sites of attachment of laminated slender crystals that were easily distinguished from macrophage surface structures. Receptors appeared most abundant on cytoplasmic veils and pseudopods and in the perinuclear region of macrophages minimally spread over the coverslip. Further macrophage spreading was associated with lighter receptor staining.

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)

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%.

Mesenchymal hamartoma of the liver: report of a case in a 28-year-old.

A 28-year-old black woman presented with increasing abdominal girth and gross hepatomegaly. Preoperative investigations demonstrated an enlarging vascular tumor involving the right lobe of the liver. A right hepatic trisegmentectomy was performed, and histologic examination of the tissue revealed a mesenchymal hamartoma, a tumor usually presenting in early childhood. This case is unique because the patient is 22 years beyond the average presenting age, and is the oldest patient reported.

Malignant rhabdoid tumor of the central nervous system.

Originally described and most frequently reported in association with the kidney, the malignant rhabdoid tumor (MRT) is a highly aggressive neoplasm with distinctive morphologic features. Extrarenal sites reported for this neoplasm include the liver, thymus, and various soft tissue sites. Young infants are affected with rare exceptions. We report the case of a 3-month-old boy who presented with hyperirritability and increasing head size over several weeks. The patient died following a two-week hospital stay marked by development of seizures, paralysis, and apnea. At autopsy, significant findings were limited to the central nervous system. The subarachnoid space contained neoplasm throughout, with multiple areas of parenchymal invasion. A predominating intraparenchymal mass was present in the inferior cerebellum contiguous with the neoplasm in the subarachnoid space and probably represented the site of origin. Microscopically, the neoplasm was composed of a highly cellular monomorphic population of polygonal cells with roughly ovoid vesicular nuclei and conspicuous nucleoli. Variable amounts of cytoplasm were present, and many cells contained a single, well-demarcated eosinophilic hyaline globule adjacent to the nucleus. Ultrastructurally, the cytoplasmic globules were composed of whorled aggregates of intermediate filaments. Immunoperoxidase studies confirmed that the filaments were composed, at least in part, of vimentin. The morphologic and immunohistochemical features are diagnostic of MRT, an entity of unknown histogenesis that has not been reported previously as a primary neoplasm of the CNS.

Characterization of a cell line derived from rhabdoid tumor of kidney.

Rhabdoid tumor of kidney (RTK) is a rare, highly malignant childhood neoplasm of uncertain histogenesis. Several recent studies have described considerable histochemical heterogeneity among cases of RTK, with confusing combinations of epithelial, mesenchymal, myogenous, and neuroepithelial markers in some tumors. The present study characterizes the histology, ultrastructure, histochemistry, cytogenetics, and oncogene expression in a cell line derived from RTK. The surgical specimen, nude mouse xenograft, and cell cultures demonstrated characteristic intermediate filament whorls by electron microscopy and expressed vimentin (diffusely) and cytokeratin (focally, in hyaline cytoplasmic inclusions) without detectable desmin, Thy-1, or epithelial membrane antigen. S-100 protein was absent in the surgical specimen and heterotransplant, and was seen very weakly and focally in the cell cultures. Light microscopic features of cultures were unchanged by several compounds (tissue plasminogen activator, nerve growth factor, cyclic adenosine monophosphate) which induce differentiation of some other pediatric neoplasms. The growth factor requirements of RTK cultures indicate a cell with mesenchymal features. Insulin-like growth factor-2 mRNA was detected in the RTK and in three Wilms' tumors also studied. Unlike most Wilms' tumors, RTK expresses the c-myc rather than the N-myc oncogene.

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.

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.