EXPERIMENTAL
AND
MOLZCULAR
Ultrastructure
PATHOLOGY
of Wilms’
A. OLUFEMI Depurtments
of Pathology
Received
24,
and
May
WILLIAMS
Surgery,
35-47
Tumor
( 1976)
(Nephroblastoma)
AND 0. 0. AJAYI
University of Ibadan lbadan, Nigeria
13, 1975;
and
in revised
and
form1 July
’
University
College
Hospital,
30, 1975
The ultr~tructura1 features of two cases of well-differentiated nephrobIastoma in Nigerian children are described. There is evidence of tubule formation morphologically similar to proximal or distal tubules of the metanephros. There is disparity between the development of microvilli and the cytoplasmic organelles. Occasional cilia or ciliary buds are seen projecting into the patent lumina of the tubules or invaginated into cytoplasmic vacuoles. Isolated particles with viral morphology, measuring between 900 and 1050A, are seen in a few cells. Some of the epithelial cells have moderate amounts of tonofibrils. The present study lends support to the concept that Wilms’ tumor is derived from metanephric blastema. The possible role of oncogenic viruses in the pathogenesis of Wilms’ tumor requires further studies.
INTRODUCTION The vast majority of TViIms’ tumor originates from the kidney (Klapproth, 1959; Lattimer et aE. 1958), but a few cases arising from extrarenal sites have been reported (Bhajekar et al., 1964; Malik et al., 1967; Thompson et aZ., 1973; and Tremblay, 1971). The histogenesis of this tumor has been controversial (Tremblay, 19i’l), but there is a consensus that it develops from embryonic metanephric blastema that is capable of differentiating into the various cellular constituents of the tumor (Ito and Johnson, 1969; Rousseau and Nabarra, 1974; Tremblay, 1971; Ward and Dehner, 1974). The morphologic similarities between the epithelial cells of nephoblastoma and normal developing human renal cells have been commented upon (Ito and Johnson, 1969; Tischer et al., 1966), but there are very few ultrastructural studies of this tumor to elucidate its histogenesis and subcellular structure (Balsaver et at., 1968; Xto and Johnson, 1969; Rousseau and Nabarra, 1974; Thompson et at,, 1973; Tremblay, 1971; and Ward and Dehner, 1974). In the course of studies on Burkitt’s lymphoma, we encountered two children with massive abdominal tumors who were thought to have Burkitt’s lymphoma on clinical grounds, Laparotomy revealed the presence of renal masses from which biopsies were taken for histopathologic diagnoses. Imprint cytology and phase contrast microscopy of the two specimens excluded the diagnosis of Burkitt’s lymphoma, and light microscopic examination confirmed that both cases were nephroblastomas. The ultrastructural features of these two biopsy specimens form the basis of this study.
Copyright All rights
@I 1976 by Academic Press, Inc. of reproduction in any form reserved.
36
WILLIAMS
MATERIALS
AND
AJAYI
AND METIIODS
Biopsy specimens were obtained from renal masses in two male Nigerian children, ages 5 and 7 yr. From each specimen, a portion was fixed in 10% formalin solution, embedded in paraffin, and stained with hematoxylin and eosin, phosphotungstic acid hematoxylin, and Masson’s trichrome. Another portion was cut into l-mm” portions and fixed in 3% glutaraldehyde for 1 hr in the cold, postosmicated, and embedded in Araldite. Two-micrometer thick sections were cut with glassknives and stained with toluidine blue and examined by phase and light microscopy to select different areas of the tumor for thin sectioning. Thin sections were stained with uranyl acetate and lead citrate and examined with a Zeiss EM9 electron microscope. RESULTS Light microscopic examination of both biopsies revealed well-differentiated tubular structures with areas of papillary projections and lobulated pseudoglomeruli (Figs. 1 and 2). Undifferentiated mesenchyme was scanty, but there were several small and large blood vessels within fibrous trabeculae (Fig. 1). Focal myxomatous areas were present, but striated muscle, bone, or areas of calification were not seen or demonstrable in the specimens examined. Diagnosis of welldifferentiated nephroblastoma ( Wilms’) was made on both specimens.
FIG. scanty x40.
1. Histologic mesenchymal
appearance component.
of tumor showing Note large thin
well-differentiated
blood vesselwithin
tubular fibrous
structures tissue. II
with & E,
WILMS’
TUMOR
37
FIG. 2. Histologic appearance of tumor showing tubular structures and two glomeruloid-like structures. Note the isolated nests of mesenchymal components in relation to the glomeruloid structures. H & E, X100.
Electron Microscopy Two populations of neoplastic cells were encountered, namely, epithelial cells lying freely or forming tubules and mesenchymal cells with varying degrees of subcellular differentiation. Mesenchymal cells were few but were easily distinguished from epithelial cells. Epithelial Cells Although these cells varied in their degree of subcellular differentiation (Figs. 3 and 4), they had certain features in common. Some nuclei were irregular in shape and size, and chromatin was distributed diffusely throughout the nuclei. There was marginal condensation in a few nuclei (Figs. 3 and 4), while some of the nuclei had prominent nucleoli (Fig. 3). The nuclei were large and were essentially oval to elongated, but a few showed irregular notches. The cytoplasm varied in its organelle content. In several of the cells forming tubules, the cytoplasm contained an abundance of ribosomes, few mitochondria, and short rough endoplasmic reticulum (Fig. 4). A thin but distinct basement membrane surrounded these cells (Figs. 3 and 4). The relationship of the epithelial cells to one another was characterized by close apposition of plasma membranes with numerous junctional complexes (Fig. 5). In places, they formed irregular extracellular spaces (Figs. 3, 4, and 6). Junctional complexes or terminal bars were not uncommon near luminal openings with microvilli (Figs. 5 and 7), but these
38
~71LLIAMS
AND
AJAYI
FIG. 3. Tubule formation by epithelinl cells. Note the presence of a few villi and a cilium projecting into the patent hunen. There are moderate amounts of free ribosomes and only a few organdies in the cytoplasm. fi-‘ote the thin, distinct basement membrane (arrow). T, lumen of tubule; S, extracellular space. U & L, X4250.
were more evident in cells with well developed Golgi complexes or centrioles (Figs. 5 and 7). Some cells revealed the formation of narrow or slit-like patent lumina with cilia and microvilli in varying stages of development ( Figs. 5 and 7). Some microvilli were pointed and interdigitated with opposing cells (Fig, S), forming irregular extracellular spaces. A perivascular arrangement of cells was seen in some areas (Fig. 4) and, in other areas, neoplastic cells were seen to be closely related to endothelial cells of blood vessels. Occasionally, apical tub&s and vesicles were seen in tumor cellsS and nlicrotubular structures around centrioles were also seen in some cells {Fig. 9). Tubules with thick basement membrane similar to Henle’s loops were not encountered. In both of our cases, there were individual fibrils measuring between 50 and 100 A in thickness (Fig. 8) or aggregates of fibrils in the cytoplasm which have been referred to as “tonofibrils’” (Ito and Johnson, 1969) (Figs. S-10). I n one of the cases, a few cells were filled with large numbers of these intertwining bundles of fibrils (Fig. 10). The aggregates of these bundles varied in thickness from 200 to 300 nm, and they were occasionally reIated to mitochondria (Fig. 10). The quantity of these fibrils
varied from cell to cell (Figs. Q and lo), and their relationship to cytoplasmic organelles or nuclei (Fig. 8) also varied. Membrane-bound autophagic vacuoles were present in some cells, and ciliary buds, invaginating into vacuoles, were also seen (Fig. 10). Occasional particles, measuring about 900 A with central nucleoids, were seen in some cells with large bundles of fibrils (Fig. lo), and open spheres of similar size range (900-1050 A) were also seen. Glycogen rosettes were seen in some eeffs ( Fig. 6)) and a few Iysosomaf bodies were aIso present (Fig. 6). CentrioIes were seen not unco~nmonly (Figs. 5, 7, and 9 f but appeared to be more common in cells with well-developed Golgi complexes. They did not appear to be related to cilia or cihary buds (Figs. 5 and 10).
These were predominantly cells with poorly differentiated cytoplasmic organelles. Their nudei were large and irregular, and the scanty cytoplasm con-
FIG. 4. The nuclei (N) of the epithelial cells are oval shaped and elongated. There are abundant ribasomes and very few cytoplasmic organelles. Note the thin basement membrane (arrows) and the relationship of the cells to a blood vessel. E, ~~do~el~l cell neucleus; S, extracellular space, U & L, X4400.
40
WILLIAMS
AND AJAYI
FIG. 5. Epithelial cells with junctional complexes (J) near patent lumen. Note the presence of a cilium (arrow) and several microvilli projecting into the lumen. There are short bundles of fibrils, which appear related to mitochondria. There is a centriole (C) and a poorly developed Golgi compIex (G). N, nucleus. X 16,800.
tamed small mitochondria, free ribosomes, and small amounts of short, rough endopfasmic reticulum; very rarely were Golgi complexes seen. No cilia were seen. Cell apposition was minimal, but small amounts of collagen were seen in the vicinity of some cells. Large osn~iophiIi~ granules were seen in a few cells close to bIood vesseis. DISCUSSION Enlargement of the abdomen due to malignancy in the African child may be due to Burkitt’s lymphoma, childhood lymphosarcoma, neuroblastoma, or nephroblastoma. Accurate diagnosis of each disease is a prerequisite for appropriate therapy and management. On a clinical basis, the different tumor types could be indistinguishable by the time they present for medical advice with abdominal masses.Furthermore, they occur in similar age groups (Williams, 1975). The two casesreported here are highly d~erentiated nep~~roblastolnaswith relatively scanty nlesenchymal components (Figs. I and 2). The refative scarcity of mesen-
WILMS’
TUMOR
41
chymal elements and absence of demonstrable mesodermal derivatives including muscle, bone, and cartilage may be due to sampling. The present study agrees to a large extent with the ul~astructural findings already reported by previous authors (Balsaver et al,, 1968; Ito and Johnson, 1969; Rousseau and Nabarra, 1974) but also presents certain additional features. Some ultrastructural features of epithelial components of Wilms’ tumor-forming tubules (Fig. 3) bear certain similarities to the normal developing metanephric tubule (Ito and Johnson, 1969). There is, however, a disparity between the d~erentiation of the microvilli and the cytoplasmic organelles in the neoplastic cells when compared with those of the normal developing metanephros (Xto and Johnson, 1969). A few proximal tubule-like structures were seen in both of our cases, but the majority of the tubular srtuctures were of the distal type (Fig. 3) with welldeveloped Golgi complexes (Figs. 5 and 7). We neither encountered tubular structures resembling Henle’s loops nor saw the cellular components of pseudo-
FIG. 6. Undifferentiated epithelial cells with very few orangellles, forming irregular extracellular spaces (S). Note the circular Golgi complex (arrow) and microtubular structures in its vicinity. Glycogen rosettes are seen scattered throughout the cyto$asm and fib&far structures are present in the vicinity of a nucleus (N). X25,600.
42
WILLIAMS
AND
AJAYI
FIG. 7. Epithelial cells with several junctional complexes (J). lmnen ( S). There is an abundance of fibrillary structures ( f) Note well-developed Golgi apparatus (G) near a centriole vesicular body (arrow) and several polyribosomes. X 19,500.
Microvilli project into a patent either dispersed or aggregated. (C). There is also a multi-
(Ward and Dehner, 1974). Apical glomeruli (Fig. 2) by electron microscopy dense tubules and coated vesicles that have been reported in the proximal convoluted tubules of the metanephros (Ito and Johnson, 1969) were occasionally seen in the epithelial cells (Fig. 9). Large bundles of fibrillar structures have been found in degenerating cpithelial tumor cells of nephroblastomas and referred to as “tonofilaments” (Ito and Johnson, 1969). In our cases, we only found bundles of tonofilaments in apparently viable tumor cells. Although fine intermingled filaments, without orientation, may suggest myofibrillogenesis in nephroblastomas (Tremblay, 1971), we observed similar filaments in our cases, but we were unable to identify the typical myogenic bands normally found in myocytes. The presence of cilia or ciliary buds (Fig. 10) in some of these cells having the largest amount of fibrils may perhaps mitigate against their either being of myogenic origin or showing myogenic differentiation.
WILMS’
TUMOR
43
Furthermore, the lack of parallel linear arrangement of the filaments and the absence of structures resembling “Z bands” may also be against the myogenic nature of these cells. The significance of these fibrils, however, remains poorly understood. Of possible relevance is the fact that thick and thin filaments have been described in normal renal tubular cells of the rat (Pease, 1968; Rostgaard and Thuneberg, 1969), and some of the filaments seem to be identical with actin filaments (Rostgaard and Thuneberg, 1969). Cilia formation has been reported in human embryonic mesonephros (De Martin0 and Zamboni, 1966) but not in human metanephros. However, cilia have been found to occur occasionally in Wilms’ tumor (Ito and Johnson, 1969) and in adult human and rat kidneys (Latta et al., 1961). This may be an expression of retrodifferentiation in Wilms’ tumor. Although the function of cilia is not known (De Martin0 and Zamboni, 1966; Ito and Johnson, 1969), it has been suggested that it is an evolutionary remnant that is commonly found in pronephric et al., 1961). We found tubules and excretory ducts of lower animals (Lattimer isolated cilia projecting into patent lumens of tubular structures (Fig, 3) and also ciliary buds within vacuoles in some undifferentiated epithelial cells packed
FIG. 8. Interdigitating brillary structures ( f) N, nucleus. X19,200.
and
villi forming irregular extracellular spaces. few cytoplasmic organelles. M, mitochondria;
Note abundance of fiJ, junctional complex;
FIG 9. Epithelial cells with a few projecting microvilli. Note the presence of junctional complexes (J) near the Lumen (arrows) with several microtubular structures and vesicles in their vicinity, and there are bundles of fibrillary structures tions in the cytoplasm. Note the relative absence of endoplasmic reticulum. M, mitochondria. N, nucleus. ~28,000.
( S). There are two centrioles ( f) running in different direc-
WILMS’
TUMOR
45
FIG. 10. Epithelial cell containing numerous bundles of fibrillary structures (f). These appear to be spatially related to mitochondria (M). There is a ciliary bud (B) invaginating into a vacuole, and there is an autophagic vacuole (V). Note the presence of an isolated particle (arrows) in relation to a cytoplasmic vacuole. N, nucleus. X 19,500.
with fibrils. Large groups of cilia have been observed in renal carcinoma of hamsters but not in normal hamster kidneys (Mannweiller and Bernhard, 1957). This suggests that if retrodifferentiation is associated with neoplasia, it can still evoke the development of highly differentiated structures of an atavistic nature. However, cilia have been seen in different segments of the adult rat nephron (Latta et al., 1961). Th is is indirect evidence of a common embryonic origin of different segments of the nephron. Cilia have also been found in the different cells of the mesonephric glomeruli and tubules (De Martin0 and Zamboni, 1966). The epithelial cells in which we observed cilia varied in their degrees of differentiation (Figs. 3,5, and 10). Although it has been reported that basal bodies of the cilia may be related to centrioles ( Ito and Johnson, 1969), we did not observe this in the present study. Furthermore, cilia were not seen in the vicinity of centrioles encountered in several epithelial and the few mesenchymal cells (Figs. 7 and 9). The significance and role of isolated particles, with viral morphology, in the epithelial cells are not clear (Fig. 10). There is some morphologic similarity be-
40
WILLTAMS
FIG 11. Higher Note the double x72,200.
AND
power view of particle (arrow) membrane of the particle and
AJAYI
in close prox~~lity to a cytoplasmic the central nucleoid. V, vacuole;
vacuole. f, fibrils.
tween this particle and BAI strain A virus, which has been associated with avian nephroblastoma (Dmochowski et al., 1961; Heine et al., 1962; Ishiguro et al., 1972). The isolated particles observed in our cases measured between 900 and 1050 A. Some of the particles were in the vicinity of c~~oplasmic vacuoles (Figs. 10 and 11). Although this particle is similar morphologically to the avian virusinduced nephroblastoma, it has a more developed nucleoid, which is surrounded by a double membrane (Fig, 11). We did not encounter such a wide variation in size of our particle as reported in virus-induced neproblastoma (Dmochowski et al., 1961), extracellular particles were not seen, and budding was not evident. The size of our particle, however, is within the range in size of the BAI strain A (myeloblastosis) virus which has been shown to be capable of inducing avian nephrobl~tomas. The exact identity of the particle observed in our case remains unknown. Virus-like particles have so far not been reported in human nephroblastomas, and the possible role of oncogenic agents in human nephroblastoma is not known. The relative scarcity of particles in solid tumors is not uncommon, but it is conceivable that more particles may become evident on tissue culture cell lines of this tumor as previously shown in the case of Burkitt’s lymphoma (Rabson et al., 1966). This study supports the observation that the subcellular structures of neoplastic epithelial cells of nep~oblastoma resemble essentially what has been described in the developing metanephros. ACKNOWLEDGMENTS We stance
wish to acknowledge DougIas. This study
the excellent was supported
technical by grants
help of Mr. A. A. Ilori and from the British Empire Cancer
Mrs. ConCampaign.
WILMS’ A. Olufemi Williams was a visiting Professor where the ultrastrudural studies were carried for the secretarial work.
47
TUMOR to out.
Howard We are
University grateful
College of to Miss Karen
Medicine Wallace
REFERENCES BALSAVER, A. M., GIBLEY, C. M., and TESSMER, C. F. (1968). Ultrastructural studies in Wilms’ tumor. Cancer 22, 417-427. BHAJEKAR, A. B., JOSEPH, M., and BHAT, H. S. (1964). Unattached nephroblastoma. Brit. J. Uroz. 36, 187-190. DE MARTINO, C., and ZAMBONI, L. ( 1966). A morphologic study of the mesonephros of the human embryo. J. Ultrastruct. Res. 16, 399-427 DMOCHOWS~, L, GREY, C. E., BURMESTER, B., and WALTER, W. G. (1961). Submicroscopic morphology of avian neoplasma in studies on nephroblastoma. Tex. Rep. Riot. Med. 19, 545-548. HEINE, U., DE THE, G., Isrrmuno, H., SOMMER, J. Ft. BEARD, D., and BEARD, J. W. (1962). Multiplicity of cell response to the BAI strain A (myeloblastosis) avian tumor virus. II. Nephroblastoma ( Wilms’ tumor) : Ultrastructure. J. Nat. Cancer Inst. 29, 41-53. ISHIGURO, H., BEARD, D., SOMMER, J. R., HEINE, U., DE THE, G., and BEARD, J. W. (1962). Multiplicity of cell response to the BAI strain A (myeloblastosis) avian tumor virus. I. Nephroblastoma ( Wilms’ tumor) : Gross and microscopic pathology. J. Nat. Cancer Inst. 29, 140. ITO, J., and JOHNSON, W. W. (1969). Ultrastructure of Wilms’ tumor. I. Epithel:al cell. J. Nat. Cancer Inst. 42, 77-99. KLAPPROTH, H. J. ( 1959). Wilms’ tumor-a report of 45 cases and an analysis of 1351 cases reported in the world literature for 1949-1958. J. Ural. 81, 633-648. LAI-IYA, H., MAINSBACH, A. B., and MADDEN, S. C. (1961). Cilia in different segments of the rat nephron. J. Biophys. Biochenz. CytoZ. 11, 248-252. LATTIMER, J. K., MELICOW, M. M., and USON, A. C. (1958). Wilms’ tumor-a report of 71 cases. J. Ural. 80, 401416. MALIK, T. K., MALIK, G. B., and BIESH, G. (1967). Retroperitoneal teratoma with nephroblastic tissue as the main component. ht. Surg. 47, 246-249. MANNWEILLER, K., and BERNHARD, W. J. ( 1957). Recherches ultrastructurales sur une tumeur renale experimentale du hamster. J. Ultrustruct. Res. I, 158-169. PEASE, D. C. (1968). Myoid features of renal corpuscles and tubules. j. Ultrastruct. Res. 23, 394-308. RABSON, A. S., O’CONNOR, G. T., BARON, S., WHANG, J. J., and LEGALLAIS, F. Y. (1966). Morphologic cytogenetic and virologic studies in vitro of a malignant lymphoma from an African child. Int. J. Cancer 1, 89-108. ROSTGAARD, J., and TFKJNEBERG, L. ( 1969). Electron microscopic evidence suggesting a contractile system in the base of the tubular cells of rat kidney. J. Ultrastruct. Res. 29, 570-576. ROUSSEAU, M. F., and NABARRA, B. (1974). Tumeurs embryonanaires du rein. Ultrastructure due nephroblastome. Virclwws Arch. A 363, 149-162. TH.OMPSON, M. R., EMANUEL, I. G., CAMPBELL, M. S., and ZACHARY, R. B. ( 1973). EXtrarenal Wilms tumor, J. Pediut. Surg. 8, 37-41. TREMBLAY, M. (1971). Ukrastructure of a Wilms’ tumor and myogenesis: J. Pathol. 105, 269-277. WARD, S. P., and DEHNER, L. P. ( 1974). Sacrococcygeal teratoma with nephroblastoma ( Wihns tumor). Cancer 33, 1355-1363. WILLIAMS, A. 0. (1975). Tumors of childhood in Ibadan, Nigeria. Cancer 36, 370379.
AND
MOLZCULAR
Ultrastructure
PATHOLOGY
of Wilms’
A. OLUFEMI Depurtments
of Pathology
Received
24,
and
May
WILLIAMS
Surgery,
35-47
Tumor
( 1976)
(Nephroblastoma)
AND 0. 0. AJAYI
University of Ibadan lbadan, Nigeria
13, 1975;
and
in revised
and
form1 July
’
University
College
Hospital,
30, 1975
The ultr~tructura1 features of two cases of well-differentiated nephrobIastoma in Nigerian children are described. There is evidence of tubule formation morphologically similar to proximal or distal tubules of the metanephros. There is disparity between the development of microvilli and the cytoplasmic organelles. Occasional cilia or ciliary buds are seen projecting into the patent lumina of the tubules or invaginated into cytoplasmic vacuoles. Isolated particles with viral morphology, measuring between 900 and 1050A, are seen in a few cells. Some of the epithelial cells have moderate amounts of tonofibrils. The present study lends support to the concept that Wilms’ tumor is derived from metanephric blastema. The possible role of oncogenic viruses in the pathogenesis of Wilms’ tumor requires further studies.
INTRODUCTION The vast majority of TViIms’ tumor originates from the kidney (Klapproth, 1959; Lattimer et aE. 1958), but a few cases arising from extrarenal sites have been reported (Bhajekar et al., 1964; Malik et al., 1967; Thompson et aZ., 1973; and Tremblay, 1971). The histogenesis of this tumor has been controversial (Tremblay, 19i’l), but there is a consensus that it develops from embryonic metanephric blastema that is capable of differentiating into the various cellular constituents of the tumor (Ito and Johnson, 1969; Rousseau and Nabarra, 1974; Tremblay, 1971; Ward and Dehner, 1974). The morphologic similarities between the epithelial cells of nephoblastoma and normal developing human renal cells have been commented upon (Ito and Johnson, 1969; Tischer et al., 1966), but there are very few ultrastructural studies of this tumor to elucidate its histogenesis and subcellular structure (Balsaver et at., 1968; Xto and Johnson, 1969; Rousseau and Nabarra, 1974; Thompson et at,, 1973; Tremblay, 1971; and Ward and Dehner, 1974). In the course of studies on Burkitt’s lymphoma, we encountered two children with massive abdominal tumors who were thought to have Burkitt’s lymphoma on clinical grounds, Laparotomy revealed the presence of renal masses from which biopsies were taken for histopathologic diagnoses. Imprint cytology and phase contrast microscopy of the two specimens excluded the diagnosis of Burkitt’s lymphoma, and light microscopic examination confirmed that both cases were nephroblastomas. The ultrastructural features of these two biopsy specimens form the basis of this study.
Copyright All rights
@I 1976 by Academic Press, Inc. of reproduction in any form reserved.
36
WILLIAMS
MATERIALS
AND
AJAYI
AND METIIODS
Biopsy specimens were obtained from renal masses in two male Nigerian children, ages 5 and 7 yr. From each specimen, a portion was fixed in 10% formalin solution, embedded in paraffin, and stained with hematoxylin and eosin, phosphotungstic acid hematoxylin, and Masson’s trichrome. Another portion was cut into l-mm” portions and fixed in 3% glutaraldehyde for 1 hr in the cold, postosmicated, and embedded in Araldite. Two-micrometer thick sections were cut with glassknives and stained with toluidine blue and examined by phase and light microscopy to select different areas of the tumor for thin sectioning. Thin sections were stained with uranyl acetate and lead citrate and examined with a Zeiss EM9 electron microscope. RESULTS Light microscopic examination of both biopsies revealed well-differentiated tubular structures with areas of papillary projections and lobulated pseudoglomeruli (Figs. 1 and 2). Undifferentiated mesenchyme was scanty, but there were several small and large blood vessels within fibrous trabeculae (Fig. 1). Focal myxomatous areas were present, but striated muscle, bone, or areas of calification were not seen or demonstrable in the specimens examined. Diagnosis of welldifferentiated nephroblastoma ( Wilms’) was made on both specimens.
FIG. scanty x40.
1. Histologic mesenchymal
appearance component.
of tumor showing Note large thin
well-differentiated
blood vesselwithin
tubular fibrous
structures tissue. II
with & E,
WILMS’
TUMOR
37
FIG. 2. Histologic appearance of tumor showing tubular structures and two glomeruloid-like structures. Note the isolated nests of mesenchymal components in relation to the glomeruloid structures. H & E, X100.
Electron Microscopy Two populations of neoplastic cells were encountered, namely, epithelial cells lying freely or forming tubules and mesenchymal cells with varying degrees of subcellular differentiation. Mesenchymal cells were few but were easily distinguished from epithelial cells. Epithelial Cells Although these cells varied in their degree of subcellular differentiation (Figs. 3 and 4), they had certain features in common. Some nuclei were irregular in shape and size, and chromatin was distributed diffusely throughout the nuclei. There was marginal condensation in a few nuclei (Figs. 3 and 4), while some of the nuclei had prominent nucleoli (Fig. 3). The nuclei were large and were essentially oval to elongated, but a few showed irregular notches. The cytoplasm varied in its organelle content. In several of the cells forming tubules, the cytoplasm contained an abundance of ribosomes, few mitochondria, and short rough endoplasmic reticulum (Fig. 4). A thin but distinct basement membrane surrounded these cells (Figs. 3 and 4). The relationship of the epithelial cells to one another was characterized by close apposition of plasma membranes with numerous junctional complexes (Fig. 5). In places, they formed irregular extracellular spaces (Figs. 3, 4, and 6). Junctional complexes or terminal bars were not uncommon near luminal openings with microvilli (Figs. 5 and 7), but these
38
~71LLIAMS
AND
AJAYI
FIG. 3. Tubule formation by epithelinl cells. Note the presence of a few villi and a cilium projecting into the patent hunen. There are moderate amounts of free ribosomes and only a few organdies in the cytoplasm. fi-‘ote the thin, distinct basement membrane (arrow). T, lumen of tubule; S, extracellular space. U & L, X4250.
were more evident in cells with well developed Golgi complexes or centrioles (Figs. 5 and 7). Some cells revealed the formation of narrow or slit-like patent lumina with cilia and microvilli in varying stages of development ( Figs. 5 and 7). Some microvilli were pointed and interdigitated with opposing cells (Fig, S), forming irregular extracellular spaces. A perivascular arrangement of cells was seen in some areas (Fig. 4) and, in other areas, neoplastic cells were seen to be closely related to endothelial cells of blood vessels. Occasionally, apical tub&s and vesicles were seen in tumor cellsS and nlicrotubular structures around centrioles were also seen in some cells {Fig. 9). Tubules with thick basement membrane similar to Henle’s loops were not encountered. In both of our cases, there were individual fibrils measuring between 50 and 100 A in thickness (Fig. 8) or aggregates of fibrils in the cytoplasm which have been referred to as “tonofibrils’” (Ito and Johnson, 1969) (Figs. S-10). I n one of the cases, a few cells were filled with large numbers of these intertwining bundles of fibrils (Fig. 10). The aggregates of these bundles varied in thickness from 200 to 300 nm, and they were occasionally reIated to mitochondria (Fig. 10). The quantity of these fibrils
varied from cell to cell (Figs. Q and lo), and their relationship to cytoplasmic organelles or nuclei (Fig. 8) also varied. Membrane-bound autophagic vacuoles were present in some cells, and ciliary buds, invaginating into vacuoles, were also seen (Fig. 10). Occasional particles, measuring about 900 A with central nucleoids, were seen in some cells with large bundles of fibrils (Fig. lo), and open spheres of similar size range (900-1050 A) were also seen. Glycogen rosettes were seen in some eeffs ( Fig. 6)) and a few Iysosomaf bodies were aIso present (Fig. 6). CentrioIes were seen not unco~nmonly (Figs. 5, 7, and 9 f but appeared to be more common in cells with well-developed Golgi complexes. They did not appear to be related to cilia or cihary buds (Figs. 5 and 10).
These were predominantly cells with poorly differentiated cytoplasmic organelles. Their nudei were large and irregular, and the scanty cytoplasm con-
FIG. 4. The nuclei (N) of the epithelial cells are oval shaped and elongated. There are abundant ribasomes and very few cytoplasmic organelles. Note the thin basement membrane (arrows) and the relationship of the cells to a blood vessel. E, ~~do~el~l cell neucleus; S, extracellular space, U & L, X4400.
40
WILLIAMS
AND AJAYI
FIG. 5. Epithelial cells with junctional complexes (J) near patent lumen. Note the presence of a cilium (arrow) and several microvilli projecting into the lumen. There are short bundles of fibrils, which appear related to mitochondria. There is a centriole (C) and a poorly developed Golgi compIex (G). N, nucleus. X 16,800.
tamed small mitochondria, free ribosomes, and small amounts of short, rough endopfasmic reticulum; very rarely were Golgi complexes seen. No cilia were seen. Cell apposition was minimal, but small amounts of collagen were seen in the vicinity of some cells. Large osn~iophiIi~ granules were seen in a few cells close to bIood vesseis. DISCUSSION Enlargement of the abdomen due to malignancy in the African child may be due to Burkitt’s lymphoma, childhood lymphosarcoma, neuroblastoma, or nephroblastoma. Accurate diagnosis of each disease is a prerequisite for appropriate therapy and management. On a clinical basis, the different tumor types could be indistinguishable by the time they present for medical advice with abdominal masses.Furthermore, they occur in similar age groups (Williams, 1975). The two casesreported here are highly d~erentiated nep~~roblastolnaswith relatively scanty nlesenchymal components (Figs. I and 2). The refative scarcity of mesen-
WILMS’
TUMOR
41
chymal elements and absence of demonstrable mesodermal derivatives including muscle, bone, and cartilage may be due to sampling. The present study agrees to a large extent with the ul~astructural findings already reported by previous authors (Balsaver et al,, 1968; Ito and Johnson, 1969; Rousseau and Nabarra, 1974) but also presents certain additional features. Some ultrastructural features of epithelial components of Wilms’ tumor-forming tubules (Fig. 3) bear certain similarities to the normal developing metanephric tubule (Ito and Johnson, 1969). There is, however, a disparity between the d~erentiation of the microvilli and the cytoplasmic organelles in the neoplastic cells when compared with those of the normal developing metanephros (Xto and Johnson, 1969). A few proximal tubule-like structures were seen in both of our cases, but the majority of the tubular srtuctures were of the distal type (Fig. 3) with welldeveloped Golgi complexes (Figs. 5 and 7). We neither encountered tubular structures resembling Henle’s loops nor saw the cellular components of pseudo-
FIG. 6. Undifferentiated epithelial cells with very few orangellles, forming irregular extracellular spaces (S). Note the circular Golgi complex (arrow) and microtubular structures in its vicinity. Glycogen rosettes are seen scattered throughout the cyto$asm and fib&far structures are present in the vicinity of a nucleus (N). X25,600.
42
WILLIAMS
AND
AJAYI
FIG. 7. Epithelial cells with several junctional complexes (J). lmnen ( S). There is an abundance of fibrillary structures ( f) Note well-developed Golgi apparatus (G) near a centriole vesicular body (arrow) and several polyribosomes. X 19,500.
Microvilli project into a patent either dispersed or aggregated. (C). There is also a multi-
(Ward and Dehner, 1974). Apical glomeruli (Fig. 2) by electron microscopy dense tubules and coated vesicles that have been reported in the proximal convoluted tubules of the metanephros (Ito and Johnson, 1969) were occasionally seen in the epithelial cells (Fig. 9). Large bundles of fibrillar structures have been found in degenerating cpithelial tumor cells of nephroblastomas and referred to as “tonofilaments” (Ito and Johnson, 1969). In our cases, we only found bundles of tonofilaments in apparently viable tumor cells. Although fine intermingled filaments, without orientation, may suggest myofibrillogenesis in nephroblastomas (Tremblay, 1971), we observed similar filaments in our cases, but we were unable to identify the typical myogenic bands normally found in myocytes. The presence of cilia or ciliary buds (Fig. 10) in some of these cells having the largest amount of fibrils may perhaps mitigate against their either being of myogenic origin or showing myogenic differentiation.
WILMS’
TUMOR
43
Furthermore, the lack of parallel linear arrangement of the filaments and the absence of structures resembling “Z bands” may also be against the myogenic nature of these cells. The significance of these fibrils, however, remains poorly understood. Of possible relevance is the fact that thick and thin filaments have been described in normal renal tubular cells of the rat (Pease, 1968; Rostgaard and Thuneberg, 1969), and some of the filaments seem to be identical with actin filaments (Rostgaard and Thuneberg, 1969). Cilia formation has been reported in human embryonic mesonephros (De Martin0 and Zamboni, 1966) but not in human metanephros. However, cilia have been found to occur occasionally in Wilms’ tumor (Ito and Johnson, 1969) and in adult human and rat kidneys (Latta et al., 1961). This may be an expression of retrodifferentiation in Wilms’ tumor. Although the function of cilia is not known (De Martin0 and Zamboni, 1966; Ito and Johnson, 1969), it has been suggested that it is an evolutionary remnant that is commonly found in pronephric et al., 1961). We found tubules and excretory ducts of lower animals (Lattimer isolated cilia projecting into patent lumens of tubular structures (Fig, 3) and also ciliary buds within vacuoles in some undifferentiated epithelial cells packed
FIG. 8. Interdigitating brillary structures ( f) N, nucleus. X19,200.
and
villi forming irregular extracellular spaces. few cytoplasmic organelles. M, mitochondria;
Note abundance of fiJ, junctional complex;
FIG 9. Epithelial cells with a few projecting microvilli. Note the presence of junctional complexes (J) near the Lumen (arrows) with several microtubular structures and vesicles in their vicinity, and there are bundles of fibrillary structures tions in the cytoplasm. Note the relative absence of endoplasmic reticulum. M, mitochondria. N, nucleus. ~28,000.
( S). There are two centrioles ( f) running in different direc-
WILMS’
TUMOR
45
FIG. 10. Epithelial cell containing numerous bundles of fibrillary structures (f). These appear to be spatially related to mitochondria (M). There is a ciliary bud (B) invaginating into a vacuole, and there is an autophagic vacuole (V). Note the presence of an isolated particle (arrows) in relation to a cytoplasmic vacuole. N, nucleus. X 19,500.
with fibrils. Large groups of cilia have been observed in renal carcinoma of hamsters but not in normal hamster kidneys (Mannweiller and Bernhard, 1957). This suggests that if retrodifferentiation is associated with neoplasia, it can still evoke the development of highly differentiated structures of an atavistic nature. However, cilia have been seen in different segments of the adult rat nephron (Latta et al., 1961). Th is is indirect evidence of a common embryonic origin of different segments of the nephron. Cilia have also been found in the different cells of the mesonephric glomeruli and tubules (De Martin0 and Zamboni, 1966). The epithelial cells in which we observed cilia varied in their degrees of differentiation (Figs. 3,5, and 10). Although it has been reported that basal bodies of the cilia may be related to centrioles ( Ito and Johnson, 1969), we did not observe this in the present study. Furthermore, cilia were not seen in the vicinity of centrioles encountered in several epithelial and the few mesenchymal cells (Figs. 7 and 9). The significance and role of isolated particles, with viral morphology, in the epithelial cells are not clear (Fig. 10). There is some morphologic similarity be-
40
WILLTAMS
FIG 11. Higher Note the double x72,200.
AND
power view of particle (arrow) membrane of the particle and
AJAYI
in close prox~~lity to a cytoplasmic the central nucleoid. V, vacuole;
vacuole. f, fibrils.
tween this particle and BAI strain A virus, which has been associated with avian nephroblastoma (Dmochowski et al., 1961; Heine et al., 1962; Ishiguro et al., 1972). The isolated particles observed in our cases measured between 900 and 1050 A. Some of the particles were in the vicinity of c~~oplasmic vacuoles (Figs. 10 and 11). Although this particle is similar morphologically to the avian virusinduced nephroblastoma, it has a more developed nucleoid, which is surrounded by a double membrane (Fig, 11). We did not encounter such a wide variation in size of our particle as reported in virus-induced neproblastoma (Dmochowski et al., 1961), extracellular particles were not seen, and budding was not evident. The size of our particle, however, is within the range in size of the BAI strain A (myeloblastosis) virus which has been shown to be capable of inducing avian nephrobl~tomas. The exact identity of the particle observed in our case remains unknown. Virus-like particles have so far not been reported in human nephroblastomas, and the possible role of oncogenic agents in human nephroblastoma is not known. The relative scarcity of particles in solid tumors is not uncommon, but it is conceivable that more particles may become evident on tissue culture cell lines of this tumor as previously shown in the case of Burkitt’s lymphoma (Rabson et al., 1966). This study supports the observation that the subcellular structures of neoplastic epithelial cells of nep~oblastoma resemble essentially what has been described in the developing metanephros. ACKNOWLEDGMENTS We stance
wish to acknowledge DougIas. This study
the excellent was supported
technical by grants
help of Mr. A. A. Ilori and from the British Empire Cancer
Mrs. ConCampaign.
WILMS’ A. Olufemi Williams was a visiting Professor where the ultrastrudural studies were carried for the secretarial work.
47
TUMOR to out.
Howard We are
University grateful
College of to Miss Karen
Medicine Wallace
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