A to a distinctive appendiceal neoplasm

Transcription

1 ULTRASTRUCTURE OF THE GOBLET CELL TYPE OF ADENOCARCINOID OF THE APPENDIX PHILIP H. COOPER, MAJOR, MC, USAR, AND RAPHAEL. WARKEL, LTC, MC, USAR Two examples of the goblet cell form of adenocarcinoid were studied with electron microscopy. Mucin was present in the form of large and small vacuoles or small patches of denser mucin granules. A small population of cells had a complement of organelles similar to the more obvious goblet cells but contained little or no identifiable product. Enterochromaffin cells were similar to those seen in normal midgut and in cotlventional carcinoids of midgut derivation. Enterochromaffin granules and much were not mixed with one another. Cell types transitional between mucinous and enterochromaffin were not found. Although study of plasmalemmae was hindered to a degree by artifacts due to tangential sectioning and, possibly, insufficiently rapid fixation, we favor the interpretation that the goblet cell type of adenocarcinoid is composed of two separate classes of cells. Cancer 42: , TTENTION HAS RECENTLY BEEN DRAWN A to a distinctive appendiceal neoplasm composed of nests of cells that resemble intestinal goblet ~ells.~,,~~ The nests frequently contain argyrophilic and argentaffin cells. These features, in combination with the anatomic site and organization, have resulted in the use of the terms mucinous carcinoid and goblet cell carcinoid to designate this tumor. In a recent report2s we described a large series of these neoplasms and recognized a tubular variant. The term adenocarcinoid was suggested for this neoplastic spectrum. In the present report we describe the ultrastructural appearance of two examples of the goblet cell type of adenocarcinoid. We also explore the problem as to whether or not the same neoplastic cell contains both mucin and enterochromaffin granules. MATERIALS AND METHODS Clinical histories, paraffin blocks, and formalin-fixed segments of appendix were avail- From the Department of Skin and Gastrointestinal Pathology, Armed Forces Institute of Pathology, Washington, D. C. Address for reprints: Philip H. Cooper, Major, MC, USAR, Department of Skin and Gastrointestinal Pathology, Arme d Forces Institute of Pathology, Washington, D. C The opinions and assertions contained herein are the private views of the authors and are not to be construed as official or as reflecting the views of the Department of the Army or the Department of Defense. Accepted for publication March 1, X/78/1200/2687 $ American Cancer Society 2687 able from two patients. Some of the transverse cut surfaces that were created during the initial gross examinations showed mural thickening. One millimeter transverse slices that included those abnormal cut surfaces were obtained and divided into blocks. The blocks were postfixed with osmium tetroxide, dehydrated, and embedded in an epoxy resin, One micron sections of the plastic-embedded tissue were stained with toluidene blue. Sections for electron microscopy were stained with uranyl acetate and lead citrate. RESULTS Two male patients, aged 51 and 65, underwent appendectomy because of a diagnosis of acute appendicitis. That diagnosis was confirmed by examination of the resected appendices. In addition a diagnosis of adenocarcinoma was made in one specimen and the terminology mucinous carcinoid was suggested for a neoplasm found in the other. One patient received no further therapy and was well two years later. The other was further treated with resection of the distal ileum and proximal colon. He died six weeks later with generalized peritonitis. The light microscopic appearance of the two neoplasms conformed to prior descrip- tion~*~~,~~ and was representative of our recently reported series (Fig. 1).28 By electron microscopy, nearly all of the epithelial nests contained mu&-producing cells. Large vacuoles occupied most of the cytoplasm of many

2 2688 CANCER December 1978 Vol. 42 FIG. 1. Nests primarily composed of goblet cells are present in the vicinity of normal mucosal crypts (toluidine blue, ~410, AFIP Neg ). of these cells and consisted of rounded, confluent granules, a few of which were partially surrounded by a delicate, smooth membrane (Fig. 2). The electron-density of the granules varied from slight to moderate, and the ap- pearance was flocculent or granular. Small amounts of cytoplasm that were not occupied by mucin contained extensive, serpiginous arrays of rough endoplasmic reticulum (RER), abundant free ribosomes, and a few mitochondria. Golgi apparatus were difficult to identify with certainty. Some cells, rather than being distended with mucin, contained one or more smaller, sharply circumscribed patches of confluent, pale mucin granules surrounded by cytoplasm rich in RER and free ribosomes (Fig. 3). A few cells contained groups of rounded mucin granules that were smaller and denser than those described above and that ranged in diameter from 100 to 600 nm (Fig. 4). Rarely these granules were partially surrounded by a smooth, delicate membrane that was closely applied to the core and they were frequently closely associated with small amounts of paler, flocculent mucin similar to that seen in well-formed vacuoles. Occasional cells (Figs. 2, 3) contained few, if any, recognizable granules but did contain RER and free ribosomes similar to those seen in mucinproducing cells. Approximately three-fourths of the neoplastic cell nests examined in a given section included sharply defined cytoplasmic areas that contained highly electron-dense, pleo- FIG. 2. A large vacuole of mucin distends a neoplastic cell. A lucent, enterochromaffin granule (EG)-containing area is sharply outlined (X6800, AFIP Neg ).

3 No. 6 GOBLET CELL ADENOCARCINOID * Cooper and Warkel 2689 FIG. 3. Some cells contain small vacuoles. EG are present between a nucleus and the stroma (~6600, AFIP Neg ). morphic enterochromaffin granules (EG) (Figs. 2, 3, 5, 6). The granules ranged in size from 100 to 425 nm and had an encircling membrane that was separated from the dense core by a narrow, lucent space of uniform width. Associated structures consisted of scattered short, parallel arrays of RER, free ribosomes, small golgi apparatus, poorly organized tracts of filaments, and structures that possibly represented microtubules (Fig. 5). In many cases the remaining cytoplasm was electron-lucent. This lucency, in contrast with adjacent denser cytoplasm, helped to emphasize the sharp circumscription of the cyto- plasmic zones that contained EG. In most examples, a portion of the granular zone abutted the fibromuscular stroma (Figs. 5, 6). In only a distinct minority of the zones was an approximately centrally placed nucleus visible, and, in such cases, a collection of granules was usually present between the nucleus and the edge of the cell nest (Fig. 3). Many EG-containing areas could not definitely be associated with a particular nucleus in the vicinity. Such areas were regarded as representing cell processes cut in various planes (Figs. 2, 6). An attempt was made to document plas- FIG. 4. A small number of mucin-producing cells contain a moderately dense form of much granule (X 15500, AFIP Neg ).

4 2690 CANCER December 1978 Vol. 42 FIG. 5. An EG-containing area is situated next to stroma (X 14000, AFIP Neg ). malemmae between cells, in particular, cells that were apparently of different types. A study of the epithelial-stromal interface illustrates one problem we encounted. In some fields a clearly visible peripheral cell membrane was closely applied to a single thin layer of basal lamina (Fig. 7) or to a longitudinally sectioned bundle of collagen fibrils. In other areas, however, collagen fibrils approached cell nests obliquely and appeared to overlap the peripheral extent of the epithelial cytoplasm (Fig. 8). In these situations a plasmalemma was not visible. A similar variation in clarity of cell membranes was found within the cell nests. Lengthy segments of relatively straight plasmalemmae were difficult to find and many images indicated complex, folded, and interdigitated cell shapes (Fig. 9). It was not usually possible to trace completely the plasmalemma of a given cell. Plasma membranes were frequently not visible between EG-containing cytoplasm and the cytoplasm ascribed to mucin-producing cells. In some of these situations, EG appeared to protrude slightly into a neighboring vacuole of mucin without an intervening plasmalemma (Fig. 8). However, as described above, the two types of cytoplasm contrasted with one another due to their different density and complement of organelles. Hence when a sharp con- FIG. 6. A cell process contains EG and is sharply outlined by adjacent dense cytoplasm. A segment of plasma membrane can be seen near the top of the image. An extracel- Mar bundle of collagen is present within an epithelial nest (X 15500, AFIP Neg ).

5 No. 6 GOBLET CELL ADENOCARCINOID. Cooper and Wurkel 2691 trast between the two was found, we gained the distinct impression that we were seeing the border between two cells (Figs. 2, 5, 6). In some of these fields, short segments of plasma membrane could be seen (Fig. 6). Further search yielded good examples of plasmalemmae between mucinous and EGcontaining cytoplasm (Figs. 10, 11). EG were not found within vacuoles of mucin or mixed with any of the forms of mucin granule. Additional observations included the following: very rare Paneth cells at the periphery of cell nests (Fig. 12); desmosomes between epithelial cells of similar and different types; bundles of tonofilaments within neoplastic cells, most of which were attached to desmosomes; and the suggestion of incipient gland lumens where several plasmalemmal folds interdigitated and a few desmosomes were present in the vicinity. DISCUSSION This study confirmed and defined in greater detail the constituents of the cell nests in the goblet cell type of adenocarcinoid. Cells distended with mucus, with nuclei pushed to a basal or peripheral position, may be termed goblet ~ ells.~*~*~~ Distinctly apical surfaces for these goblet cells were not defined, however, since well-developed lumens were not formed. Other examples of this neoplasm have shown lumens by light microscopy. Another way in which the neoplastic goblet cell deviated from the normal was in the apparent, nearly complete absence of nonmucus-filled Golgi apparatus adjacent to nuclei. Some Golgi membranes are probably occupied in partially surrounding mucin granules, and details of others were perhaps lost because of insufficiently rapid initial fixation. Golgi membranes may be poorly developed in these cells. A significant population of cells was identified that contained mucin but that could not be called goblet cells. They had one or a few small vacuoles, or lacked vacuoles but contained patches of smaller, denser mucin granules. Review of paraffin-28 and plastic-embedded sections identified cells which correlated with these mucin-producing types. It cannot be stated from our observations whether or not these cells become goblet cells as may be the case in the normal colonic ~rypt.~,~~,~ It is possible that this population, with less cytoplasmic maturation, is relatively stable in terms of differentiation and that it harbors FIG. 7. Peripheral cell membranes and basal lamina are clearly depicted in this field. Desmosomes with attached tonofilaments are present (X29100, AFIP Neg ). FIG. 8. Stromal collagen fibrils overlap and obscure the peripheral cell membrane. EG appear to protrude slightly :Into an adjacent vacuole oi -mucin (X29100, AFIP Neg ).

6 2692 CANCER December 1978 Vol. 42 FIG. 9. Examination of plasma membranes indicates complex cell shapes with short, interdigitating processes ( ~ 1800, 2 AFIP Neg ). some increased potential for aggressive behavior. Cells with similar features are found by electron microscopy in conventional colonic adenomas and adenocar~inomas.~,'~*'~ Moreover, mature-appearing goblet cells were in the distinct minority in most of the metastases from goblet cell adenocarcinoids described in a recent report.28 The type of enterochromaffin cell was uniform throughout both neoplasms studied. The ultrastructural features were similar to those seen in most conventional carcinoids of the appendi~~.'~ or elsewhere in midgut derivatives. 3,4,21329 A s is the case with many carcinoids, the neoplastic cells recapitulated, both histochemicallyz8 and ultrastructurally, the features of normal enterochromaffin cells located in the crypts of the segment of intestine in which the neoplasm is 10cated.~ They were consistently situated basally in the cell nest and had processes that extended either circumferentially or, less commonly, a short distance toward the center of the cell nest. The overall organization of the cellular nests, including the peripherally located Paneth cells, was reminiscent of the normal crypt. Because of the processes, the number of enterochromaffin cells per cell nest may be overestimated when viewed by light microscopy. Furthermore, in a given plane of section, EG may not be found in some cell nests by electron microscopy. In some examples, multiple deeper sections revealed one or two EG-containing processes. Nevertheless, based on light and electron microscopic observations, it appears likely that some nests do not contain enterochromaffin cells. A major focus of interest in this study was the question of whether or not there were cells in this neoplasm which both produced mucus and contained EG. The existence of such transitional cells would be of considerable interest since it could be construed as evidence in favor of an endodermal and against a neuroectodermal origin for enterochromaffin cells and ordinary carcinoids. Neither origin is proven. Reports have appeared, however, that describe gastrointestinal neoplasms with features that suggest transitional forms between conventional carcinoid and the usual adenocarcinoma~l,2~ ,199z3-z5~z7 Two of these reports describe some ultrastructural aspects of appendiceal neoplasms that appear related to more typical examples of the goblet cell' and tubularz3 forms of adenocarcinoid. There has been a trend toward the interpretation that such neoplasms represent evidence in favor of a common, endodermally derived progenitor for both the carcinoid and conventional carcinoma. Indeed, electron micrographs have been published for which the claim is made that mucin and EG are present in the same cell.'," On the other hand, intermediate forms were not found in a gastric carcinoid that contained well-formed goblet cell-lined luminae within the carcinoidal Our interpretation is similar to that last cited.4 Both by light microscopy, using a combined silver-mucin stain,z8 and by electron microscopy, mucin and EG were not found to be mixed with one another. The cytoplasmic constituents of the two apparent cell types were distinctly different, and, because of differences in cytoplasmic density, the two cell types frequently contrasted with each other. There were some similarities between the

7 No. 6 GOBLET CELL ADENOCARCINOID. Cooper and Warkel 2693 FIGS. 10 (top)- 11 (bottom). Plasma membranes separate mucin-producing and enterochromaffin cells (x and 27800, respectively, AFIP Negs and 13). dense form of mucin granule and the EG. However, the former were somewhat larger, were consistently rounded or oval and not pleomorphic, were usually less dense than EG, and were only occasionally bounded by a membrane. Moreover, they were associated with paler, flocculent mucin granules, and were not mixed with the pleomorphic, highlydense, membrane-bound structures with characteristics of EG. Nevertheless, a significant problem encountered in this material was a failure to consistently document distinct cell membranes between cells that appeared to be of different types. We believe that this may be explained, in part, by the infrequency with which cell margins are likely to be sectioned in a plane close to perpendicular in this particular neoplasm. The cell nests are small and may be approximately spherical in some instances. Complex folds and interdigitations of cell membranes were documented. Many cell membranes are likely to be sectioned in a tangential fashion. Such sectioning would seem to be a reasonable explanation for appearances such as that in Fig. 8 in which collagen fibrils overlap the peripheral layers of neoplastic cell cytoplasm. A membrane separating the cytoplasm from the stroma is not visible and this is felt to be due to tangential sectioning. The same artifact

8 2694 CANCER December 1978 Vol. 42 FIG. 12. A Paneth cell is present at the periphery of a cell nest (~6600, AFIP Neg ). most likely also explains the apparent absence of membranes between the mucinous and EGcontaining cytoplasm in the same figure. In ideal sections, segments of plasmalemmae between neoplastic cells and between the neoplasm and stroma were clearly visible. Indeed, examples of unequivocal cell membranes between mucin- and EG-containing cytoplasm were documented. Insufficiently rapid initial fixation probably also hindered more extensive documentation of cell membranes. Although our observations are not con- clusive, we believe they favor the interpretation that there are two separate major cell populations within the two examples of goblet cell adenocarcinoid analyzed in this study. These are mucinous-including the goblet cell-and enterochromaffin. It is certainly possible that we have overlooked rare intermediate cell types. We are not convinced, however, by previously published histochemical and ultrastructural evidence that the controversial cell containing both mucin and neurosecretory-like enterochromaffin granules has been successfully documented. REFERENCES 1. Abt, A. B., and Carter, S. L.: Goblet cell carcinoid of the appendix. An ultrastructural and histochemical study. Arch. Pathol. Lab. Med. 100: , Bates, H. R., Jr., and Belter, L. F.: Composite carcinoid tumor (argentaffinoma-adenocarcinoma) of the colon: report of two cases. Dis. Colon Rectum 10: , Black, W. C., 111: Enterochromaffin cell types and corresponding carcinoid tumors. Lab. Invest. 19: , Black, W. C., and Haffner, H. E.: Diffuse hyperplasia of gastric argyrophil cells and multiple carcinoid tumors. An historical and ultrastructural study. Cancer 21: , Fisher, E. R., and Sharkey, D. A,: The ultrastructure of colonic polyps and cancer with special reference to the epithelial inclusion bodies of Leuchtenberger. Cancer 15: , Florey, H. W.: Electron microscopic observations on goblet cells of the rat s colon. (2. J. Exp. Physiol. 45: , Freeman, J. A,: Goblet cell fine structure. Anat. Rec. 154: , Gagni., F., Fortin, P., Dufour, V., and Delage, C.: Tumeurs de l appendice associant des caractkres hisrologiques de carcinoide et d adknocarcinome. Ann. Anat. Pathol. 14: , Gibbs, N. M.: The histogenesis of carcinoid tumours of the rectum.]. Clin. Pathol. 16: , Goldenberg, D. M., and Fisher, E. R.: Histogenetic relationship between carcinoids and mucin-secreting carcinomas of colon as revealed by heterotransplantation. Br. J. Cancer 24: , Hernandez, F. J., and Fernandez, B. B.: Mucussecreting colonic carcinoid tumors: light- and electronmicroscopic study of three cases. Dis. Colon Rrctum 17: , Hernandez, F. J.. and Reid, J. D.: Mixed carcinoid and mucus-secreting intestinal tumors. Arch. Pathol. 88: , Horn, R. C., Jr.: Carcinoid tumors of the colon and rectum. Cancer 2: , 1949.

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