Histochemical and Immunohistochemical Characterization of ... · Herman Yeger,2 Reuben Baumal, Dennis Bailey, Gladys Pawlin, and M. James Phillips Department of Pathology, The Hospital

[CANCER RESEARCH 45, 2350-2357, May 1985]

Histochemical and Immunohistochemical Characterization of SurgicallyResected and Heterotransplanted Wilms' Tumor1

Herman Yeger,2 Reuben Baumal, Dennis Bailey, Gladys Pawlin, and M. James Phillips

Department of Pathology, The Hospital lor Sick Children and the Toronto General Hospital, Toronto, Canada M5G 1X8

ABSTRACT

Nine surgically resected Wilms' tumors (WIT) and nude mouse

heterotransplants from one WIT were studied by histochemistryand immunohistochemistry. Histochemistry showed acid phos-phatase in all cells, while alkaline phosphatase and -y-glutamyl

transpeptidase were present in only some tubules. Using immunohistochemistry, antibodies to the intermediate filaments cyto-keratin and vimentin distinguished tubular epithelium and mes-

enchyme, respectively. WIT tubules were also identified usingantibody against a structural component (epithelial membraneantigen) and a secretory product (uromucoid) associated withdistal convoluted tubules of normal kidney. Basement membranesurrounding the tubules of WIT was demonstrated using antibody to type IV collagen plus laminin. Different blastema sub-

populations were negative or stained positively with antibodiesto cytokeratin and vimentin. Production of basement membraneby blastema was also shown. Fetal antigen expression in WITwas examined using the monoclonal P1153/3 and J5 antibodies.The blastema and tubules of WIT were strongly stained by PI153/3, which did not label normal adult kidney, and weaklystained by J5, which strongly labeled glomeruli and proximalconvoluted tubules of normal kidney. These studies show thatWIT blastema is heterogeneous in intermediate filament subtypes, while WIT tubules more closely resemble distal thanproximal convoluted tubules of adult kidneys but also retainexpression of fetal antigens.

INTRODUCTION

WIT3 or nephroblastoma, a solid tumor of childhood, has a

distinctive histological appearance, consisting of blastema, tubules, and mesenchyme (4, 24). In addition, WIT variants havebeen described which contain blastema and tubules, tubulesalone, and blastema associated with differentiated structuressuch as skeletal muscle (4). The tubules of WIT have beenshown to retain morphological and biochemical similarities toproximal and distal convoluted tubules, including production ofBB antigen and uromucoid, respectively (39). Early attempts toprepare antibodies to the different cell types of WIT have metwith some success (6, 33), but a more definitive study has notappeared. Thus, it is still unknown whether unique, stage-specific

antigens are present in WIT, as reported in mouse and humanteratocarcinomas (2).

1This work was supported by the National Cancer Institute of Canada.2To whom requests for reprints should be addressed, at Department of Pathol

ogy, The Hospital for Sick Children, 555 University Avenue, Toronto, Ontario,Canada M5G 1X8.

3The abbreviations used are: WIT, Wilms' tumor; ALP, alkaline phosphatase;

ACP, acid phosphatase; i-GT, 7-glutamyl transpeptidase; i.d., intradermal(ly); EMA,epithelial membrane antigen; MBCK, Mallory body cytokeratin; PAP, peroxi-dase:antiperoxidase; CALLA, common acute lymphoblastic leukemia antigen.

Received 7/23/84; revised 12/5/84; accepted 1/28/85.

WIT can be successfully passaged as heterotransplants innude mice (27, 31, 40), and histological examination has shownthat the nude mouse tumors closely resemble the surgicallyresected specimens. In the present study, 9 surgically resectedWIT and the nude mouse heterotransplants derived from one ofthem were studied by histochemistry and immunohistochemistryto identify the different tumor cell types present.

MATERIALS AND METHODS

Samples of surgically resected WIT were taken for morphological(histology and ultrastructure), histochemical, immunohistochemical, andnude mouse heterotransplantation studies.

Morphology. Histological evaluation was performed as described inour previous paper (40).

Histochemistry. Unfixed, S-^m cryostat sections of 9 surgically re

sected WIT and nude mouse heterotransplants of one of these wereexamined for ALP, ACP, and y-GT (29). Normal kidney was used as a

control because these enzymes are located in different parts of thenephron.

Immunohistochemistry. Surgically resected and heterotransplantedWIT were stained by immunofluorescence and immunoperoxidase techniques using: (a) polyclonal antibodies to the intermediate filament cytokeratin (22), EMA (34), and uromucoid (Tamm-Horsfall secretory glyco-

protein) (39) to label epithelial cells; (ft) polyclonal antibody to the intermediate filament vimentin (8) to label mesenchymal cells; and (c) monoclonal antibodies, P1153/3 and J5, in an attempt to label blastema cells.In addition, basement membrane material in WIT was labeled usingpolyclonal antibody to a mixture of type IV collagen plus laminin.

Mallory bodies were extracted from cirrhotic human liver, and a rabbitantibody to MBCK was produced (22) which reacted with various typesof epithelial cells, including proximal and distal convoluted tubules andcollecting ducts of normal human kidney. Goat antibody to EMA (SeraLaboratory, Crawley Down, Sussex, England), which reacted with thesurface glycoproteins of secretory cells, was prepared against an organicsolvent extract of milk fat globule membranes (34). Rabbit antibody touromucoid, specific for the Tamm-Horsfall protein which is synthesized

by epithelial cells of the loop of Henle and distal convoluted tubule, waspurchased from Cappel Laboratories, Inc. (Malvern, PA). Rabbit antibodyto basement membrane was prepared against a pepsin digest of placentacontaining type IV collagen (Sigma Chemical Co., St. Louis, MO) (10).Immunoblotting showed that the anti-basement membrane antibody was

directed against type IV collagen plus laminin (37). This antibody stainedglomerular capillary and tubular basement membranes of normal kidney.A rabbit antibody to vimentin, which reacted with mesenchymal cells,was produced by immunization with vimentin extracted from a culturedline of human embryonic lung fibroblasts (8, 19). Mouse monoclonalantibody J5, which recognizes the CALLA on lymphocytes, was purchased from Coulter Diagnostics (Hialeah, FL), while PI 153/3, whichrecognizes an antigenic determinant on the human neuroblastoma cellline IMR-5, fetal brain cells, and normal and leukemic pre-B- and matureB-lymphocytes (21), was purchased from Bethesda Research Labora

tories (Gaithersburg, MD).All antibodies prepared by us were produced by giving injections to

New Zealand White rabbits s.c. or i.d. of 1 mg of antigen in Freund's

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WIT IMMUNOHISTOCHEMISTRY

Table 1Histochemical staining ot WIT, WIT-1 heterotransplant, and normal kidney lor ALP, ACP, and y-GT

Staining was membranous and cytoplasmic for ALP, diffuse throughout the cytoplasm for ACP, and of luminal contents and at the apices of tubules for -y-GT.

Histochemical staining" for

ALP y-GT ACP

Blastema Tubules Mesenchyme

SpecimenWIT-1WIT-5WIT-6WIT-7WIT-8WIT-9WIT-10WIT-11WIT-12WIT-1 hetero

transplant

Normal kidney

Absent

Absent

Absent

PCT0 DOT

Blastema Tubules Mesenchyme Blastema Tubules Mesenchyme

Absent

Absent

Absent Absent

Absent

Absent

Glomeruli Stroma PCT DCT Glomeruli Stroma PCT DOT Glomeruli Stroma

* Staining intensity was graded negative (-), weak (+), moderate (++), and strong (+++).b Only one-half of the tubules were ALP positive.c PCT, proximal convoluted tubules; DCT, distal convoluted tubules.

complete adjuvant. Booster injections were given at 2-week intervals,

and sera were collected after 6 weeks.Immunofluorescence and Immunoperoxidase Labeling. Two label

ing methods were used, depending on the ability of the various antibodiesto recognize antigenic determinants in formalin-fixed or frozen tissuesections. For rabbit anti-MBCK and goat anti-EMA antibodies, tissueswere fixed in 10% buffered formalin and embedded in paraffin. Five-urn

sections were deparaffinized, rehydrated, and briefly digested with pepsin to expose antigenic determinants (18). Immunolabeling was performed by the PAP method (20). Normal rabbit and goat sera were usedas negative controls. In the case of rabbit antibodies to uromucoid,basement membrane (type IV collagen plus laminin), and vimentin andthe mouse monoclonal antibodies, J5 and PI 153/3, tissues were snap-frozen in isopentaneiliquid nitrogen, cut into 5-^m cryostat sections, air

dried, fixed in acetone for 15 min, air dried again, and immediately stainedor stored at -20Â°C. For the rabbit antibodies, an indirect immunofluo-

rescence method was applied, using fluorescein isothiocyanate-labeledgoat anti-rabbit immunoglobulin (Miles-Yeda. Ltd., Rehovot, Israel) as

the secondary reagent (19). For the mouse monoclonal antibodies, anindirect immunoperoxidase technique was applied, using peroxidase-conjugated Ffab'fe goat anti-mouse immunoglobulin as the secondary

reagent. The method used was that described by Habeshaw ef a/. (11)for labeling of lymphocyte antigens. Positive controls for the monoclonalantibodies were normal kidney and a neuroblastoma. Negative controlswere performed by substituting normal mouse serum for the monoclonalantibodies.

Heterotransplantation of WIT. The procedures for preparing tumorcells for injection of WIT into nude mice and assessment of tumor growthhave been described in the preceding paper (40).

RESULTS

The results of histochemical studies of surgically resectedWIT, nude mouse heterotransplants of WIT-1, and normal kidney

are shown in Table 1.ALP, ACP, and 7-GT were present in proximal and distal

convoluted tubules of normal kidney (Figs. 1 to 3). ALP and 7-

GT staining was stronger in proximal than in distal convolutedtubules, whereas ACP staining was stronger in distal tubules.Up to one-half of the tubules in some cases of WIT and nudemouse heterotransplants of WIT showed weak-to-moderate

reactivity for ALP and 7-GT, but blastema was negative (Figs. 1to 3). Overall, ALP and 7-GT reactivities were much stronger in

normal kidney than in tumor. In addition, tubules and blastemashowed staining for ACP in most cases. These studies suggestthat some of the tubules of WIT have undergone partial differentiation and resemble proximal convoluted tubules, while othersresemble distal convoluted tubules.

The results of immunohistochemical studies of surgically resected WIT, nude mouse heterotransplants of WIT-1, normal

kidney, and renal cell carcinoma are shown in Tables 2 to 4.A PAP technique was used to label the MBCK and EMA in

tubular epithelium. In normal kidney, antibody to MBCK stronglystained the cytoplasm of collecting ducts, distal convolutedtubules, and portions of proximal convoluted tubules, whereasantibody to EMA labeled the apical portions of distal convolutedtubules (data not shown). In the case of WIT and nude mouseheterotransplants of WIT-1, both antibodies labeled the tubules

in most cases. Staining was heaviest at the apices and lateralcell margins (probably corresponding to desmosomal contactsbetween cells) and weakest at the basal aspects (Figs. 4 and 5).Since antibody to MBCK and EMA labeled mainly distal convoluted tubules and collecting ducts of normal kidney, these resultssuggest that the tubules of WIT resembled these types of tubulesin most cases. No labeling of mesenchyme was seen with theseantibodies, whereas blastema was either negative or positive indiscrete cytoplasmic areas, especially with the antibody toMBCK. Antibody to uromucoid produced diffuse cytoplasmicstaining of the tubules of WIT and nude mouse heterotransplantsof WIT-1, as detected by indirect immunofluorescence (Fig. 6).

In normal kidney, uromucoid is produced by epithelial cells of theascending portion of the loop of Henle and distal convolutedtubules (15). Therefore, positive staining of the tubules of WITsuggested that they were similar to this portion of the nephron.The presence of a basement membrane around the tubules ofWIT and nude mouse heterotransplants of WIT-1 was confirmed

by indirect immunofluorescence using antibody to basementmembrane components (type IV collagen plus laminin) (Fig. 7).In addition, deposits of basement membrane material were detected between blastema cells, suggesting that blastema was


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Table2Immunoperoxidase staining of WIT, WIT-1 heterotransplant, and normal kidney with polyclonal antibodies to MBCK and EMA

Antibody to MBCK was used at a 1:200 dilution and stained the apices and Å“il junctions of tubules and focal areas of blastema. Antibody to EMA was used at a1:400 dilution and stained the apices of tubules and luminal contents.

Immunoperoxidase staining8 using polyclonal antibodiestoSpecimenWIT-1WIT-5WIT-6WIT-7WIT-8WIT-9WIT-1

0WIT-11WIT-12WIT-1

hetero-transplantMBCKBlastema

Tubules MesenchymeBlastema+

++ â€”â€”++â€”â€”++

Absent+AbsentAbsent

-Absent+++ â€”â€”+++ â€”â€”Absent

Absent++ â€”â€”+

++ â€” â€”EMATubules

Mesenchyme+++

â€”+++â€”AbsentAbsent+

-+++â€”+â€”â€”â€”+

â€”+++â€”

Normal kidneyPCT* DOT Glomeruli Stroma PCT DCT Glomeruli Stroma

a Staining intensity was graded negative (-), weak (+), moderate (++), and strong (+++)â€¢6 Only host stroma present.c PCT. proximal convoluted tubules; DCT, distal convoluted tubules.

TaWe3

Immunoperoxidase staining of WIT, adult tubular WIT, renal cell carcinoma, and normal kidney using monoclonal antibodies J5 and PI 153/3

J5 and PI 153/3 were used at recommended concentrations (usually 1:10). Staining with both monoclonals was at the apices of tubules, luminal contents and thecytoplasm, and plasma membranes of blastema cells.

Immunoperoxidase staining8 using monoclonal antibodies to

J5 P1153/3

Blastema

SpecimenWIT-1WIT-5WIT-6WIT-7WIT-8WIT-9WIT-10WIT-11WIT-12Adult tubular WIT

Renal cell carcinoma

Absent

Absent

Tubules

Absent

Mesenchyme Blastema

Absent

Absent

Tumor cells

Tubules Mesenchyme

Absent

Tumor cells

Normal kidneyPCT6 DCT Glomeruli Sfroma PCT DCT Glomeruli Stroma

8 Staining intensity was graded negative (-), weak (+), moderate (++), and strong (+++).6 PCT, proximal convoluted tubules; DCT, distal convoluted tubules.

Table 4

Immunohistochemical staining of WIT and normal kidney with polyclonal antibodies to uromucoid, vimentin, and basement membrane

Surgically resected WIT Normal kidney

Type of staining Blastema Tubules Mesenchyme PCTÂ« DCT Glomeruli StromaAntiuromucoid (1:15)6

Antivimentin (1:20)Anti-BM (1:40)

8 PCT, proximal convoluted tubules; DCT, distal convoluted tubules; BM, basement membrane.0 Numbers in parentheses, dilution used.c Staining intensity was graded as negative (-). weak (+), moderate (++), and strong (+++).

capable of producing basement membrane.To label mesenchyme, antibody to vimentin was used by

indirect immunofluorescence. This antibody stained endothelialand epithelial cells of glomeruli and stromal cells around thetubules of normal kidney. In WIT and nude mouse heterotrans-

plants of WIT-1, antibody to vimentin also labeled the mesenchy-

mal component. Tubules were negative, while some of theblastema cells were stained in a pattern resembling that seenwith the antibody to basement membrane (Fig. 8).

In addition to anti-vimentin antibody, blastema was labeled


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with 2 additional sets of antibodies, (a) Using antibody to MBCKin a PAP reaction, the blastema immediately peripheral to tubulesor just beginning to form tubules stained positively (Fig. 4). A!!other areas of blastema were negative. Therefore, this antibodyappeared to recognize those blastema cells which were committed to tubular epithelial differentiation, (b) Monoclonal antibodies,PI 153/3 and J5, were used in an indirect immunoperoxidasereaction. In normal kidney, PI 153/3 did not stain glomeruli ortubules, while both the blastema and tubules of WIT stainedpositively (Fig. 9). The adult tubular WIT also showed strongstaining throughout the cytoplasm of tubular epithelium whichwas heaviest at cell apices. Therefore, the blastema and especially the tubular cells retained expression of fetal antigens duringmorphological and functional maturation. In normal kidney, however, J5 stained glomeruli and proximal but not distal convolutedtubules (Fig. 10). The blastema and tubules of WIT stained onlyweakly with this antibody or were negative. However, in the caseof an adult tubular WIT, J5 strongly stained the epithelium. Thus,J5 recognized antigenic determinants on tubular epithelium ofnormal kidney and on an adult WIT, but not on childhood WIT,suggesting that these determinants were expressed later indevelopment. In general, J5 or PI 153/3 did not stain stromalcells of WIT, but in a small number of cells, weak staining wasobserved. In a case of renal cell carcinoma, the tumor cellsstained weakly with J5 and PI 153/3 (not shown). Since renalcell carcinoma is thought to arise from the proximal convolutedtubule (39), the J5 staining was not surprising, while reactivitywith PI 153/3 indicated retention of fetal antigens and a histo-

genetic link to WIT.

DISCUSSION

The present study has utilized histochemical and immunohis-

tochemical methods to shed light on the different cell components of WIT. The presence and degree of reactivity of ALP (38),ACP, and 7-GT, as detected by histochemical staining in tubules

of WIT, suggest a similarity to the distal convoluted tubule portionof the nepnron. Histochemical staining for ALP reactivity wasdemonstrated in a small portion of tubules of both primary WITand a WIT heterotransplant. The degree of staining in the majorityof WIT was considerably less than that found in the proximalconvoluted tubules of normal kidney and more comparable tothat seen in distal convoluted tubules. Waghe and Kumar (38)also found little ALP reactivity in WIT and showed a much simplerisoenzyme pattern for ALP and nonspecific esterase in WITextracts. Thus, it would be interesting to know whether alteredisoenzymes occur during the development of WIT and are linkedin some way to specific genetic abnormalities associated withthis tumor (25).

The results of our study and other reports (1, 9) have shownthat the tubules of WIT contain cytokeratin and EMA, secreteuromucoid, and synthesize basement membrane material (3, 26,36). Cytokeratin and EMA are produced mainly by collectingducts and distal convoluted tubules of normal kidney, whileuromucoid is synthesized only by the ascending portion ofHenle's loop and distal convoluted tubules. Therefore, these

structural and secretory components are satisfactory for demonstrating the similarity of the tubules of WIT to the distal tubularsystem of normal kidney. HolthÃ¶feref al. (13), using monoclonalantibodies to cytokeratins and a rabbit anti-vimentin antibody,

described stage-dependent developmental patterns in the

expression of intermediate filaments in fetal and adult humankidneys. Undifferentiated mesenchyme of fetal kidney expressedvimentin, while collecting ducts, which produce cytokeratin inadult kidneys, also showed a transient expression of vimentin.Developing proximal and distal tubular cells expressed cytokeratins, but certain cytokeratin staining patterns were lost duringnephrogenesis. Thus, dynamic changes in intermediate filamentcomposition and organization were observed. Differentiating WITmight be expected to show a similar complexity in intermediatefilament expression.

Recently, Altmannsberger et al. (1) reported that the blastemaof WIT stained for cytokeratin and vimentin, as detected byimmunofluorescence. Such double expression of these 2 typesof intermediate filaments by a single cell type occurs only rarelyin vivo (28). In addition to staining tubules of WIT, the antibodyto MBCK used in this study labeled blastema cells which wereadjacent to and undergoing differentiation into tubules. Thisfinding indicates that epithelial differentiation is accompanied bythe turning on of genes which code for intermediate filamentexpression. This type of gene activation is also found when germcells, which do not express intermediate filaments, develop intopreimplantation embryos (17). Vimentin staining, albeit weak,was found in blastema cells in our study, suggesting that someof these cells retained mesenchymal characteristics. Moreover,vimentin expression appeared to be retained as that of cytokeratin was initiated (1). It is therefore clear from our studies andthose of others (1) that the heterogeneity in intermediate filamentexpression in blastema reflects a multipotential capacity fordifferentiation by these cells. Moreover, it is likely that the majority of blastema cells are partially differentiated, since thesecells have rudimentary intercellular junctions and produce basement membrane components.

Monoclonal antibody J5, which detects CALLA, has beenreported to stain fetal tubules as well as proximal convolutedtubules and glomeruli of fully developed human kidney (30). Inchildhood WIT, there was only slight labeling of blastema andtubules by J5, but in a case of adult tubular WIT, there wasstrong staining of tubular epithelium. This suggests that tubulesare more differentiated in adult than in childhood WIT. Therefore,the antigenic determinants recognized by J5 appeared to relateto the maturation of proximal convoluted tubules. In contrast,tubular epithelium of normal kidney was not labeled using PI153/3, whereas blastema cells and tubules of WIT were stronglylabeled. Since the determinants recognized by PI 153/3 (previously noted on neuroblastoma cells, B-lymphocytes, and fetal

brain) are probably of fetal origin, both the blastema and tubulesshare fetal antigens. Fetal antigens are frequently expressed ina variety of tumor cell types and are much less common innormal tissues (16). For example, a-fetoprotein is often found in

high concentrations on hepatomas and carcinoembryonic antigen on colonie carcinomas. Thus, in the case of WIT, fetalantigenic expression by tubular epithelium appears to be retainedwhile the cells undergo morphological and functional maturation.

No immunological marker is available to distinguish blastemacells of WIT from their differentiated derivatives. Platt ef al. (30)reported that BA-1 monoclonal antibody, which identifies a sur

face antigen expressed by granulocytes and by normal andmalignant B-lymphocytes at various stages of differentiation,

also reacts with cells in the kidney at the earliest stages of


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nephron development. Preliminary data suggest that BA-1 alsolabels the blastema cells of WIT.4 Another method for distinguish

ing blastema cells and differentiated derivatives of WIT might beto examine the surface glycoproteins of cells. This could beaccomplished using lectins, which have been shown to staindifferent parts of the nephron of normal kidney (14). It has beenclaimed that benign and malignant tumors can be differentiatedon the basis of lectin binding (23). Preliminary studies in otherlaboratories (12) are under way to determine whether lectins ofknown specificity will discriminate among the various types ofWIT and selectively stain the blastema cells in this tumor.

It has been postulated that the blastema of WIT is generatedfrom metanephric mesenchyme through a process of inductionsimilar to that which occurs in the developing nephron (4, 7, 24,32). However, in the case of WIT, further development of blastema is blocked, leading only to the formation of disorganized,nonfunctional tubules and abortive glomeruli. The factors thatgovern this limited differentiation of blastema are unknown.Some of the controlling events may be at the gene level, specifically in association with a recognized aberration in chromosome11 (11p13) (25). By being able to identify the various cell typesin WIT, it should now be possible to induce tumor cell differentiation using proven differentiating agents (5, 35). Such studies arein progress.

ACKNOWLEDGMENTS

The assistance of the Medical Publications Department, The Hospital for SickChildren, Toronto, Canada, is acknowledged.

REFERENCES

1. Altmannsberger, M., Osborn, M., Schafer, H., Schauer, A., and Weber, K.Distinction of nephroblastomas from other childhood tumors using antibodiesto intermediate filaments. Virchows Arch. B Cell Pathol., 45: 113-124,1984.

2. Andrews, P. W., Damjanov, I., Simon, D., Banting, G. S., Carlin, C., Dracopoli,N. C., and Fogh, J. Pluripotent embryonal carcinoma clones derived from thehuman teratocarcinoma cell line Tera-2: differentiation in vivo and in vitro. Lab.Invest., 50: 147-162, 1984.

3. Bachmann, S., Kriz, W., KÃ¼hn,C., and Franke, W. W. Differentiation of celltypes in the mammalian kidney by immunofluorescence microscopy usingantibodies to intermediate filament proteins and desmoplakins. Histochemistry,77: 365-394, 1983.

4. Beckwith. J. B. Wilms' tumor and other renal tumors of childhood: a selectivereview from the National Wilms' Tumor Study Pathology Center. Hum. Pathol.,

74:481-492,1983.5. Bloch, A. Induced cell differentiation in cancer therapy. Cancer Treat. Rep.,

68: 199-205: 1984.6. Burtin, P., and Gendron, M. C. A tumor-associated antigen in human nephro

blastomas. Proc. Nati. Acad. Sci. USA, 70: 2051-2054, 1973.7. Chatten, J. Epithelial differentiation in Wilms' tumour: a clinicopathologic ap

praisal. Perspect. Pediatr. Pathol., 3: 225-254, 1976.8. Franke. W. W., Schmid, E., Winter, S., Osbom, M., and Weber, K. Widespread

occurrence of intermediate-sized filaments of the vimentin-type in culturedcells from diverse vertebrates. Exp. Cell Res., 723: 25-46, 1979.

9. Franklin, W. A., and Ringus, J. C. Basement membrane antigen in Wilms'

tumor. Lab. Invest., 44: 375-380, 1981.10. Glanville, R. W.. Rauter, A., and Fietzek, P. P. Isolation and characterization

of a native placental basement-membrane collagen and its component achains. Eur. J. Biochem., 95: 383-389, 1979.

11. Habeshaw, J. A., Bailey, D., Stansfeld, A. G., and Greaves, M. F. The cellularcontent of non Hodgkin lymphomas: a comprehensive analysis using monoclonal antibodies and other surface marker techniques. Br. J. Cancer, 47: 327-

4 H. Yeger, R. Baumal, D. Bailey, G. Pawlin. and M. J. Phillips, unpublished data.

351,1983.12. Hennigar, R. A., Spicer, S. S., Sens, D. A., Newman, V., and Garvin, A. J.

Lectin histochemistry of nephroblastoma (abstract). Lab. Invest., 50: 25A,1984.

13. HolthÃ¶fer, H., Miettinen, A., Lehto, V. P., Lehtonen, E., and Virtanen, I.Expression of vimentin and cytokeratin types of intermediate filament proteinsin developing and adult human kidneys. Lab. Invest., 50: 552-559, 1984.

14. HolthÃ¶fer, H., Virtanen, I., Pettersson, E., TÃ¶mroth, T., Alfthan, 0., Linder, E.,and Miettinen, A. Lectins as fluorescence microscopic markers for saccharidesin the human kidney. Lab. Invest., 45: 391-399,1981.

15. Hoyer, J. R., Resnick, J. S., Michael, A. F., and Vernier, R. L. Ontogeny ofTamm-Horsfall urinary glycoprotein. Lab. Invest., 30: 757-761, 1974.

16. Ibsen, K. H., and Fishman, W. H. Developmental gene expression in cancer.Biochim. Biophys. Acta, 560: 243-280,1979.

17. Jackson, B. W., Grund, C., Schmid, E., BÃ¼rki,K., Franke, W. W., and Illmensee,K. Formation of cytoskeletal elements during mouse embryogenesis: intermediate filaments of the cytokeratin type and desmosomes in preimplantationembryos. Differentiation, 17: 161-179, 1980.

18. Kahn, H. J., Marks, A., Thorn, H., and Baumal, R. Role of antibody to S100protein in diagnostic pathology. Am. J. Clin. Pathol., 79:341-347,1983.

19. Kahn, H. J., Yeger, H., Baumal, R., Thorn, H., and Phillips, M. J. Categorizationof pediatrie neoplasms by immunostaining with antiprekeratin and antivimentinantisera. Cancer (Phil.), 57: 645-653,1983.

20. Kahn, H. J., Yeger, H., Kassim, 0., Jorgensen, A. 0., MacLennan, D. H.,Baumal, R., Smith, C. R., and Phillips, M. J. Immunohistochemical and electronmicroscopic assessment of childhood rhabdomyosarcoma. Cancer (Phila.), 57:1897-1903,1983.

21. Kennet!, R. H., and Gilbert, F. Hybrid myelomas producing antibodies againsta human neuroblastoma antigen present on fetal brain. Science (Wash. DC),203: 1120-1121, 1979.

22. Kimoff, R. J., and Huang, S. N. Immunocytochemical and immunoelectronmicroscopic studies on Mallory bodies. Lab. Invest., 45: 491-503, 1981.

23. Louis, C. J., Sztynda, T., Cheung, Z. M., and Wyllie, R. G. Lectin-bindingaffinities of human breast tumors. Cancer (Phila.), 52: 1244-1250,1983.

24. Marsden, H. B. The pathology and natural history of childhood tumours. RecentResults Cancer Res., 88:11-25,1983.

25. Matsunaga, E. Genetics of Wilms' tumor. Hum. Genet., 57; 231-246,1981.

26. Nagle, R. B., McDaniel, K. M., Clark, V. A., and Payne, C. M. The use ofantikeratin antibodies in the diagnosis of human neoplasms. Am. J. Clin.Pathol., 79:458-466, 1983.

27. Neely, J. E., Ballard, E. T., Britt, A. L., and Workman, L. Characteristics of 85pediatrie tumors heterotransplanted into nude mice. Exp. Cell. Biol. 57: 217-227, 1983.

28. Osbom, M., and Weber, K. Intermediate filaments: cell-type-specific markersin differentiation and pathology. Cell, 37: 303-306, 1982.

29. Pearse, A. G. E. Histochemistry Theoretical and Applied, Ed. 3. Edinburgh:Churchill Livingstone, 1972.

30. Platt, J. L., LeBien, T. W., and Michael, A. F. Stages of renal ontogenesisidentified by monoclonal antibodies reactive with lymphohemopoietic differentiation antigens. J. Exp. Med., 757: 155-172,1983.

31. Rousseau-Merck, M. F., Cottreau, D., and Kahn, A. Isozyme pattern in seriallyxenotransplanted childhood tumors. Cancer Res., 44; 1163-1166,1984.

32. Schmidt, D., Dickersin, G. R., Vawter, G. F., Mackay, B., and Harms, D. Wilms'

tumor: review of ultrastructure and histogeneiss. Pathobiol. Annu., 72: 281-300, 1982.

33. Seeger, R. C., Zeltzer, P. M., and Rayner, S. A. Once-neural antigen: a newneural differentiation antigen expressed by neuroblastoma, oat cell carcinoma,Wilms' tumor, and sarcoma cells. J. Immunol., 722: 1548-1555, 1979.

34. Sloane, J. P., and Ormerod, M. G. Distribution of epithelial membrane antigenin normal and neoplastic tissues and its value in diagnostic tumor pathology.Cancer (Phila.), 47: 1786-1795, 1981.

35. Speers, W. C. Conversion of malignant murine embryonal carcinomas to benignteratomas by chemical induction of differentiation in vivo. Cancer Res., 42:1843-1849,1982.

36. Sun, T. T., Shih, C. H., and Green, H. Keratin cytoskeletons in epithelial cellsof internal organs. Proc. Nati. Acad. Sci. USA, 76: 2813-2817, 1979.

37. Towbin, H., Staehelin, T., and Gordon, J. Electrophoretic transfer of proteinsfrom polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. Nati. Acad. Sci. USA, 76: 4350-4354,1979.

38. Waghe, M., and Kumar, S. Studies of hydrolytic enzymes and isoenzymes ofnormal and neoplastic childhood renal tissues and their tissue cultured cells.Br. J. Urol., 49:189-194, 1977.

39. Wallace, A. C., and Nairn, R. C. Renal tubular antigens in kidney tumors.Cancer (Phila.), 29: 977-981,1972.

40. Yeger, H., Baumal, R., Pawlin, G., and Phillips, M. J. Relationship of histologyof Wilms' tumor to growth characteristics of nude mouse heterotransplants.

Cancer Res., 45: 2340-2349,1985.


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Figs. 1 to 3. Histochemistry of WIT and normal kidney. Fig. 1. ACP reaction, showing a strong staining of tubules and glomeruli (G) in normal kidney (a; x 200) andmoderate staining of blastema (B) and strong staining of tubules (7")of WIT (b; x 400). Fig. 2. ALP reaction, showing a strong staining of proximal convoluted tubules,

weaker staining of distal convoluted tubules, and no staining of glomeruli (G) of normal kidney (a; x 200) and moderate staining of some tubules (T) but no staining ofothers in WIT. Blastema (B) is also negative except in peritubular areas (middle bottom) (b; x 400). Fig. 3. >-GT reaction, showing a strong staining of proximal convolutedtubules and weaker staining of distal convoluted tubules of normal kidney (a; x 200) and strong staining of the apices of some tubular cells (T) of WIT (Â£>).


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Figs. 4 and 5. Immunoperoxidase labeling of surgically resected WIT and WIT heterotransplant using anti-MBCK and anti-EMA antibodies. Fig. 4. Anti-MBCK antibodylabeling, showing staining of the apices and plasma membranes of tubular cells (T) of surgically resected WIT (a and inset, left top) and staining of tubules (T) but notblastema (B) of WIT heterotransplant (/>, /aft bottom), a, x 100; Ã¶,x 200. Fig. 5. Anti-EMA antibody labeling, showing staining of the apices of tubular cells (7) and theluminal contents of WIT (a, right top) and staining of apices of tubular cells (T) of WIT heterotransplant (b, right bottom), x 200.


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Figs. 6 to 8. Immunofluorescent labeling of WIT using anti-basement membrane, anti-vimentin, and anti-uromucoid (Tamm-Horsfall glycoprotein) antibodies. Fig. 6.Anti-uromucoid antibody labeling showing staining at the apices of tubules (arrowheads) but not of the blastema of WIT (right bottom), x 400. Fig. 7. Anti-basementmembrane antibody labeling, showing strong staining of the basal aspects of tubular cells and intercellular stroma of WIT-1, with no staining of blastema (a), and strongstaining of the basement membrane (arrowheads) surrounding the tubular cells in WIT-10, a monomorphous tubular tumor (b). x 400. Fig. 8. Anti-vimentin antibodylabeling, showing staining of blastema (arrowheads) and mesenchyme but not tubules of WIT. x 400.

Figs. 9 and 10. Immunoperoxidase labeling of normal kidney and WIT, using monoclonal anti-CALLA (J5) and anti-neuroblastoma (PI 153/3) antibodies. Fig. 9.Labeling with PI 153/3, showing no staining of normal kidney (a) and strong staining of the cytoplasm and plasma membranes of blastema (B) and tubules (T) of WIT (b).x 200. Fig. 10. Labeling with J5, showing strong staining of glomeruli and apices of proximal convoluted tubules of normal kidney (a) and weak staining of tubules ofWIT (b). x 200.


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1985;45:2350-2357. Cancer Res Herman Yeger, Reuben Baumal, Dennis Bailey, et al. Surgically Resected and Heterotransplanted Wilms' TumorHistochemical and Immunohistochemical Characterization of

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Histochemical and Immunohistochemical Characterization of ... · Herman Yeger,2 Reuben Baumal, Dennis Bailey, Gladys Pawlin, and M. James Phillips Department of Pathology, The Hospital