Some ultrastructural features of acinic cell carcinoma By G. D. BLOOM, B. CARLSOO
and R. HENRIKSSON* (Umea, Sweden) cell carcinoma is a rather uncommon neoplasm of salivary glands, accounting for approximately 3 per cent of all parotid gland tumours. The tumour is exceedingly rare in the minor salivary glands and the sublingual gland, and only roughly 5 per cent of the acinic cell carcinomas originate in the submandibular gland (Evans and Cruickshank, 1970; Blanck, 1974; Batsakis, 1974). Long term follow-up studies have clearly established that this tumour is to be classified as a carcinoma. It shows a rather high recurrence rate and it may either infiltrate locally or metastasize (Abrams et al., 1965 Eneroth et al. 1966). It has, however, been questioned if all acinic cell tumours should be considered malignant since many do not behave aggressively (Smith and Fechner, 1974). The histopathological picture is generally highly characteristic, and the resemblance of the tumour cells to normal parotid secretory cells is striking. In many areas the granulated cells delimit follicular compartments, forming acinar-like structures. However, histopathological features, on the basis of which the biological behaviour of the neoplasm can be prognosticated, appear to be lacking. Mitotic activity, nuclear and cellular atypias as well as growth pattern, do not seem to have any apparent significance as far as prognosis is concerned (Batsakis, 1974). On the other hand, paralysis of the facial nerve is considered indicative of a very poor prognosis (Eneroth, 1972; Eneroth and Hamberger, 1974). It was therefore considered of interest to establish if any subcellular features were characteristic and could be of aid in diagnosis with respect to degree of malignancy of these tumours. For this purpose an acinic cell carcinoma of the left parotid gland, in a 52-year-old female patient exhibiting a total facial nerve paralysis, was studied in both the light and the electron microscope. Case Report The patient ( $ 52 years of age) was admitted to the university ENTdepartment for investigation of a total left-sided facial paralysis. Over a period of 10 months, the patient had 4 attacks of partial facial paresis, all of which had shown a marked remission after 2 to 3 weeks. Two months before admission to the clinic, the patient had again noticed an onset of facial weakness, which ACINIC
. * Departments of Histology and Otolaryngology, University of Umea.
947
G. D. Bloom, B. Carlsoo and R. Henriksson did not appear to subside. In addition, for about one year, the patient had been aware of a left-sided, swollen, slightly painful retromandibular mass which had not markedly increased in size over this time period. Physical examination revealed a total facial paralysis as well as a palpable, slightly tender tumour mass, between the mastoid process and the mandible. Electromyography showed complete denervation of all three main branches of the facial nerve. Audiometry was normal bilaterally. However, no stapedius reflex could be evoked on the left side. X-ray tomography of the left facial canal showed a pathological widening of its mastoid portion. Aspiration biopsy from the tumour revealed a fairly uniform cell pattern of granulated tumour cells. Diagnosis: a probable acinic cell carcinoma. The patient underwent total parotidectomy, as well as partial resection of the mastoid process. Materials and Methods
During the operation, tissue specimens from different parts of the neoplasm were removed and cut into small pieces which were rapidly transferred to the fixatives employed. The specimens were fixed (a) for 4 hr at 4°C in 4 per cent phosphate buffered glutaraldehyde (pH 7-4), rinsed in buffer and postfixed in 1 per cent osmium tetroxide for 2 hrs. Specimens were also fixed primarily in (b) 1 per cent phosphate buffered osmium tetroxide (pH 7-4) (Millonig, 1961) for 3 hrs at 4°C. Following OsO4 fixation or postfixation the specimens were rinsed in phosphate buffer for two hours, dehydrated in graded ethanol solutions, followed by propylene oxide and were embedded in Epon. In addition specimens were (c) fixed in ice-cold 3 per cent KMnO4 (Richardson, 1966) in Krebs-Ringer phosphate buffer (pH 7-0) for 60-90 min, rinsed in Ringer solution and contrasted en bloc for 60 min in 1 per cent uranyl acetate (Hokfelt, 1968). These specimens were then dehydrated as above and embedded in Epon. Semithin and thin sections were cut on an LKB Ultrotome. The thin sections were collected on copper grids and contrasted with lead citrate or with uranyl acetate followed by lead citrate. For demonstration of periodate reactive substances at the ultrastructural level, tumour specimens were (d) fixed for 4 hrs in 2 • 5 per cent glutaraldehyde (o-i M phosphate buffer, pH 7-4). Following a two hour buffer rinse, the tissues were dehydrated in ethanol and styrene and embedded in Vestopal W. Thin sections were collected on gold grids, and treated with the periodic acid-silver (PA-CrA-silver) technique of Rambourg et al. (1969). All thin sections were examined in a Philips EM 300 electron microscope. Results
In semithin toluidine blue stained Epon sections, it is seen in the light microscope that cellular differentiation varies within different tumour areas. In certain regions the cells are pyramidal in shape and form acinarlike structures with central lumina. In these tumour cells there is present 948
Some ultrastructural features of acinic cell carcinoma a barely discernible population of discrete cytoplasmic granules, predominantly located in the apical portion of the cells. Compared to acinar cells of normal parotid tissue, the granules of the tumour cells are very inconspicuous at the light microscopical level (compare Figs, i and 2). In other tumour regions the cells form solid cell strands and are virtually agranular. In tumour specimens fixed in glutaraldehyde-OsO4 according to (a) and studied in the electron microscope a number of the granulated tumour cells display a fairly electron-lucid cytoplasm (light cells). However, cells with a more dense appearance (dark cells) are also observed. The latter are less common than the light cells and transitions from one type to the other are also encountered. Electron microscopy further reveals that the cytoplasmic granules of individual tumour cells differ markedly with respect to size, internal electron density and packing. In some cells, the membrane-bounded apical granules are loosely dispersed; they vary in size and display an electron dense, homogeneous appearance. In others, the granules are densely packed, are larger and more uniform in size and their contents consist of a finely flocculent material of rather low electron density. In numerous such granules a bipartite pattern is discerned, i.e. they exhibit a minute, central, dense core which is surrounded by a fairly broad rim of electron lucid material (Fig. 3).
FIG. I.
Semithin Epon section of normal parotid tissue, stained with toluidine blue. Numerous acinar cells are depicted, filled with large well-defined secretory granules. Light micrograph. X570
949
G. D. Bloom, B. Carlsoo and R. Henriksson
FIG. 2. Semithin Epon section of acinic cell carcinoma. In certain areas the tumour cells are arranged around lumen-like spaces (arrows), and in their supranuclear regions the cells display minute cytoplasmic granules. Light micrograph. X570
FIG. 3. Apical portions of two granulated tumour cells fixed in glutaraldehyde and postfixed in OsO«. A number of granules display a bipartite structure with a small dense core and a more lucid outer rim. Electron micrograph. x6-8oo
950
Some ultrastructural features of acinic cell carcinoma In specimens fixed in Millonig's fixative (b) tumour cell cytoplasm in general appears more homogeneous with respect to overall electron density. On the other hand, this technique shows up the cytoplasmic granules so electron lucid as to give the impression of membrane-bounded vesicles (Fig. 4). Also after fixation in KMnO4, (c), followed by en bloc staining with uranyl acetate and contrasting of the thin sections with lead citrate, the granules showed a similar 'empty' appearance. Nuclear polymorphism is not pronounced; the nuclei are generally rounded or oval in shape. They consistantly exhibit one or two conspicuous nucleoli and display numerous, rather regularly spaced, aggregates of chromatin at the nuclear margin. The cells in sections contain only few profiles of rough endoplasmic reticulum (RER), and only occasionally are the latter arranged in a regular pattern. Lysosome-like structures as well as lipid droplets are present in most cells but are rather inconspicuous. Golgi complexes, sometimes well-developed, appear in close proximity to the cell nucleus. The most striking feature of the tumour cell is the weird appearance of their mitochondria. Not only is their size increased but there is also a pronounced disorientation of their cristae (Fig. 5). The latter, instead of traversing the internal matrix of this organelle, often run parallel to the circumference thereby forming circles or whorl-like formations. In addition, portions of the internal membranes are vesiculated, thus creating a
FIG. 4. Granulated cell of acinic cell carcinoma specimen primarily fixed in OsO4-. All apical granules display an empty appearance. Electron micrograph. X4 75O
951
G. D. Bloom, B. Carlsoo and R. Henriksson picture resembling a string of empty beads. In some mitochondria, lastly, the internal compartment is completely occupied by microtubular structures (Fig. 5). Another characteristic feature of the cells in the present tumour is the smooth cell surfaces between adjacent cells. Lateral interdigitations are rare. Also the luminal cell surfaces are mostly even and only few microvilli are observed. In various regions along apposed cell borders subplasmalemmal bands of electron dense material are present in the cytoplasm of adjacent ells (Fig. 6). Furthermore, numerous intercellular attachments of desmosomal type may be seen. Applying the PA-CrA-silver technique to ultrathin sections of Vestopal embedded material, reaction products are observed over the cytoplasmic granules (Fig. 7). In addition, basement membrane structures stain well as do collagen fibres of the connective tissue. Finally, in certain tumour areas neoplastic cells are smaller in size and totally lack cytoplasmic granules (Fig. 8). These cells are rich in organelles and mitochondria especially are numerous. As in the granulated tumour cells mitochondrial structure is markedly altered. The cells also contain numerous cytoplasmic vacuoles the contents of which are considerably less electron dense than the cytoplasmic ground substance. Desmosomal structures are numerous and the subplasmalemmal bands of
FIG. 5. Basal and perinuclear region of tumour cell specimen fixed in OsO4. Numerous mitochondria are depicted which exhibit marked alterations in size and internal structure. Electron micrograph, x 11 • 900
952
Some ultrastructural features of acinic cell carcinoma
FIG. 6. Junction area between four tumour cells. Specimen fixed in OsO«. Numerous desmosomes are observed as is a long, dense subplasmalemmal band in two apposed cells. Electron micrograph, x 19-650
FIG. 7. Glutaraldehyde fixed specimen of acinic cell carcinoma, embedded in Vestopal and stained according to the Rambourg technique. No counterstain. Numerous granules in two tumour cells exhibit a strong positive reaction. Electron micrograph. X9"3oo
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G. D. Bloom, B. Carlsoo and R. Henriksson
FIG. 8. Electron micrograph of OsO4-fixed specimen from a tumour area consisting of agranulated cells. Note the smooth cell borders as well as the dense cytoplasmic bands in the contact region of two adjacent cells (arrow). Lipid droplets and lysosomal structures are also present.
X 3•800
dense material described above are also common in these cells. No cells resembling myoepithelial or striated duct cells were encountered in the present study. 954
Some ultrastructural features of acinic cell carcinoma Discussion Persistent facial nerve paralysis, caused by a malignant parotid tumour, is a serious prognostic sign (Eneroth, 1972). The incidence of facial nerve paralysis in the acinic cell carcinoma group of malignant salivary gland tumours is, however, reported to be low. Eneroth (1972), in a series of 378 patients with malignant parotid neoplasms, recorded 46 patients with spontaneous facial paralysis. Only one of these patients had an acinic cell carcinoma. In a series of 279 malignant parotid tumours recently reviewed by Conley and Hamaker (1975), 34 patients presented involvement of the facial nerve. None of the patients with acinic cell carcinoma (a total of 17) exhibited engagement of the facial nerve. The main electron microscopic findings recorded in the present tumour are in general agreement with previous studies (Kleinsasser et al., 1967; Erlandson and Tandler, 1972; Fechner et al., 1972; Kay and Schatzki, 1972). Thus the size as well as the internal density of the cytoplasmic granules varies considerably in these tumours. However, Fechner et al. (1972) described smooth cell borders without desmosomal attachments, whereas Hiibner et al. (1968) reported an abundance of desmosomes as well as peculiar, dense, band-like structures in the most peripheral cytoplasm of the cells. The latter authors also observed well-developed intercellular interdigitations between the tumour cells. The tumour cells studied in the present investigation exhibited both smooth cell surfaces and numerous desmosome-like structures as well as the subplasmalemmal material of Hiibner et al. (1968). The mitochondrial structure of previously described cells in this type of carcinoma has not been reported as abnormal. In the present case, however, gross changes were observed in this organelle. Mitochondria of abnormal appearance are otherwise a highly characteristic feature of another salivary gland tumour, i.e. the Warthin tumour (e.g. Tandler, 1966). It should also be pointed out that structural alterations of this organelle are very common in various neoplasias (Bernhard, 1969). The zymogen granules of serous acinar cells of the major human salivary glands often exhibit, at the electron microscopic level, substructures of a characteristic appearance. Thus, in the parotid and submandibular gland the granules display a bipartite structure with a relatively dense core surrounded by a more electron-lucid outer rim (Tandler and Erlandson, 1972; Riva and Riva-Testa, 1973; Riva et al., 1974). Moreover pleomorphic secretory granules have been depicted in the salivary glands of numerous mammalian species, as well as in other exocrine cell systems (for reference see Tandler and MacCallum, 1972; Borghese et al., 1974; Bloom et al., ig77). The significance of this ordered arrangement of material within salivary gland granules is unclear. As in normal parotid tissue, the granules of numerous neoplastic cells in the described acinic cell carcinoma displayed a bipartite structure after fixation in glutaraldehyde followed by 955
G. D. Bloom, B. Carlsoo and R. Henriksson osmium tetroxide. Millonig's fixative (i per cent osmium tetroxide) as employed in the present study, gave an overall satisfactory preservation of the tissue. However, the granules of the tumour cells appeared leachedout. Similar observations have been reported in a previous study on such a tumour (Erlandson and Tandler, 1972) as well as in the normal human submandibular gland (Tandler and Erlandson, 1972). Amsterdam and Schramm (1966) found that isolated rat parotid zymogen granules, exposed to osmium, release up to 75 per cent of their protein content into the fixing fluid, resulting in a collapse of the granules. According to the same authors the granules are not affected by glutaraldehyde and retain all of their protein in this fixative. It may thus be assumed that there is a marked protein leakage from the granules of the acinic tumour cells when fixed in osmium tetroxide. From their histochemical and ultrastructural features it seems reasonable to assume that the granulated cells of acinic cell carcinoma are derived from the serous acinar cells of the salivary gland secretory units. However, the origin of the small agranular cells observed in the present report is uncertain. The intercalated duct cells of human salivary glands display many submicroscopic features in common with these agranular tumour cells. The intercalated duct cells are cuboidal in shape, exhibit smooth cell borders with numerous desmosomes, a finely filamentous cytoplasm and a poorly developed rough endoplasmic reticulum (Chisholm et al. 1974; Riva et al. 1976). It should be pointed out that Abrams and co-workers (1965) in their material (77 cases) recorded 5 acinic cell carcinomas in which the main cellular constituent was of the intercalated duct cell type. In addition Hirtzler et al. (1969) described tumour cells of two acinic cell carcinomas which at the ultrastructural level correspond to intercalated duct cells. The so-called clear cell acinic cell carcinoma is proposed to originate from striated duct cells (Echevarria, 1967). No cells whatsoever with features of striated duct cells were observed in the present study. The patient reported on in the present study exhibited a total denervation of the left facial nerve. Thus, the investigated tumour showed a local, aggressive, growth pattern. Although the overall histological picture as well as the ultrastructural appearance of the cells in general is similar to that described for acinic cell carcinomas with a more benign course, a striking difference was noted with respect to the gross pathological alterations of mitochondrial fine structure. Summary
The ultrastructure of an acinic cell carcinoma, occurring in the left parotid gland of a 52-year-old woman and causing a total facial nerve paralysis, is described. Histologically the tumour consisted of numerous granulated cells arranged around lumen-like openings and resembling a 956
Some ultrastructural features of acinic cell carcinoma secretory system. Furthermore, areas with agranulated cells growing in a solid pattern were also encountered. In the electron microscope the cytoplasmic granules of the tumour cells displayed a varied appearance. Granules of a dense homogeneous type, as well as granules with a more electron lucid appearance were observed. Furthermore, numerous cytoplasmic granules displayed a bipartite structure with a dense central and a more electron lucid outer zone. In specimens primarily fixed in OsO4 or KMnO4 the granules displayed a 'leached out' appearance. The membrane-bounded granules of the tumour cells also showed a strong positive staining with the periodic acid-chromic silver technique of Rambourg et al. (1969). Other characteristic ultrastructural features of the tumour cells studied were: Smooth cell surfaces, the presence of subplasmalemmal bands of electron dense material, desmosome-like attachment areas between cells and grossly altered mitochondria. REFERENCES ABRAMS, A. M., CORNYN, J., SCOFIELD, H. H. and HANSEN, L. S. (1965) Cancer 18,
"45AMSTERDAM, A. and SCHRAMM, M. (1966) Journal of Cell Biology 29, 199. BATSAKIS, J. G. (1974) Tumors of the head and neck. The Williams & Wilkins Company, Baltimore. BERNHARD, W. (1969) In Handbook of molecular cytology. (Ed. A. Lima-De-Faria). North-Holland Publishing Company, Amsterdam, London, p . 687. BLANCK, C. (1974) Carcinoma of the Parotid Gland. Acta Universitatis Upsaliensis, Uppsala, Sweden, Diss Med. BLOOM, G. D., CARLSOO, B., DANIELSSON, A. and WAHLIN, T. (1977)
Histochemistry,
51, 261. BORGHESE, E., LAJ, M. and Di CATERINO, B. (1974) Cell Tissue Research 150, 425. CHISHOLM, D. M., WATERHOUSE, J. P., KRAUCUNAS, E. and SCIUBBA, J. J. (1974)
Cancer 34, 1631. CONLEY, J. and HAMAKER, R. C. (1975) Archives of Otolaryngology 101, 39. ECHEVARRIA, R. A. (1967) Cancer 20, 563. ENEROTH, C.-M., JACOBSSON, P. A. and BLANCK, C. (1966) Cancer 19, 1761.
— (1972) Archives of Otolaryngology 95, 300. —, and HAMBERGER, C.-A. (1974) The Laryngoscope, LXXXIV, 1732. ERLANDSON, R. A. and TANDLER, B. (1972) Archives of Pathology 93, 130. EVANS, R. W. and CRUICKSHANK, A. H. (1970) Epithelial tumours of the salivary glands. W. B. Saunders Company, Philadelphia, London, Toronto. FECHNER, R. E., BENTINCK, B. R. and ASKEW, J. B . (1972) Cancer 29, 501. HIRTZLER, R., OBERMAN, B., KULIS, M. and LJUBESIC, N. (1969) Archiv fur Klinische
und Experimentelle Ohren-, Nasen und Kehlkopfheilkunde
195, 68.
HUBNER, G., KLEIN, H. J. and KLEINSASSER, O. (1968) Virchows Archiv, Abteilung
A, Pathologie und Anatomie 345, 1. HOKFELT, T. (1968) Zeitschrift fur Zellforschung 91, 1. KAY, S. and SCHATZKI, P. F. (1972) Cancer 29, 235. KLEINSASSER, O., HUBNER, G. and KLEIN, H. J. (1967) Archiv fur Klinische und
Experimentelle Ohren-, Nasen- und Kehlkopfheilkunden MILLONIG, G. (1961) Journal of applied Physics 32,1637.
189, 33.
RAMBOURG, A., HERNANDEZ, W. and LEBLOND, C. P. (1969) Journal of Cell Biology
'
40,395-
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G. D. Bloom, B. Carlsoo and R. Henriksson RICHARDSON, K. C. (1966) Nature 210, 756. RIVA, A. and RIVA-TESTA, F. (1973) The Anatomical Record 176,149. •—, MOTTA, G. and RIVA-TESTA, F. (1974) The American Journal of Anatomy 139, 293—, TESTA-RIVA, F., DEL FIACCO, M. and LANTINI, M. S. (1976) Journal of Anatomy 122, 627. SMITH, T. E. and FECHNER, R. E. (1974) Archives of Otolaryngology 100, 324. TANDLER, B. (1966) Archives of Otolaryngology 84, 68. —, and ERLANDSON, R. A. (1972) American Journal of Anatomy 135, 419. —, and MACCALLUM, D. K. (1972) Journal of Ultrastructural Research 39, 186. Departments of Histology and Otolaryngology, University of Umea, s-901 87 Umea, Sweden.
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and R. HENRIKSSON* (Umea, Sweden) cell carcinoma is a rather uncommon neoplasm of salivary glands, accounting for approximately 3 per cent of all parotid gland tumours. The tumour is exceedingly rare in the minor salivary glands and the sublingual gland, and only roughly 5 per cent of the acinic cell carcinomas originate in the submandibular gland (Evans and Cruickshank, 1970; Blanck, 1974; Batsakis, 1974). Long term follow-up studies have clearly established that this tumour is to be classified as a carcinoma. It shows a rather high recurrence rate and it may either infiltrate locally or metastasize (Abrams et al., 1965 Eneroth et al. 1966). It has, however, been questioned if all acinic cell tumours should be considered malignant since many do not behave aggressively (Smith and Fechner, 1974). The histopathological picture is generally highly characteristic, and the resemblance of the tumour cells to normal parotid secretory cells is striking. In many areas the granulated cells delimit follicular compartments, forming acinar-like structures. However, histopathological features, on the basis of which the biological behaviour of the neoplasm can be prognosticated, appear to be lacking. Mitotic activity, nuclear and cellular atypias as well as growth pattern, do not seem to have any apparent significance as far as prognosis is concerned (Batsakis, 1974). On the other hand, paralysis of the facial nerve is considered indicative of a very poor prognosis (Eneroth, 1972; Eneroth and Hamberger, 1974). It was therefore considered of interest to establish if any subcellular features were characteristic and could be of aid in diagnosis with respect to degree of malignancy of these tumours. For this purpose an acinic cell carcinoma of the left parotid gland, in a 52-year-old female patient exhibiting a total facial nerve paralysis, was studied in both the light and the electron microscope. Case Report The patient ( $ 52 years of age) was admitted to the university ENTdepartment for investigation of a total left-sided facial paralysis. Over a period of 10 months, the patient had 4 attacks of partial facial paresis, all of which had shown a marked remission after 2 to 3 weeks. Two months before admission to the clinic, the patient had again noticed an onset of facial weakness, which ACINIC
. * Departments of Histology and Otolaryngology, University of Umea.
947
G. D. Bloom, B. Carlsoo and R. Henriksson did not appear to subside. In addition, for about one year, the patient had been aware of a left-sided, swollen, slightly painful retromandibular mass which had not markedly increased in size over this time period. Physical examination revealed a total facial paralysis as well as a palpable, slightly tender tumour mass, between the mastoid process and the mandible. Electromyography showed complete denervation of all three main branches of the facial nerve. Audiometry was normal bilaterally. However, no stapedius reflex could be evoked on the left side. X-ray tomography of the left facial canal showed a pathological widening of its mastoid portion. Aspiration biopsy from the tumour revealed a fairly uniform cell pattern of granulated tumour cells. Diagnosis: a probable acinic cell carcinoma. The patient underwent total parotidectomy, as well as partial resection of the mastoid process. Materials and Methods
During the operation, tissue specimens from different parts of the neoplasm were removed and cut into small pieces which were rapidly transferred to the fixatives employed. The specimens were fixed (a) for 4 hr at 4°C in 4 per cent phosphate buffered glutaraldehyde (pH 7-4), rinsed in buffer and postfixed in 1 per cent osmium tetroxide for 2 hrs. Specimens were also fixed primarily in (b) 1 per cent phosphate buffered osmium tetroxide (pH 7-4) (Millonig, 1961) for 3 hrs at 4°C. Following OsO4 fixation or postfixation the specimens were rinsed in phosphate buffer for two hours, dehydrated in graded ethanol solutions, followed by propylene oxide and were embedded in Epon. In addition specimens were (c) fixed in ice-cold 3 per cent KMnO4 (Richardson, 1966) in Krebs-Ringer phosphate buffer (pH 7-0) for 60-90 min, rinsed in Ringer solution and contrasted en bloc for 60 min in 1 per cent uranyl acetate (Hokfelt, 1968). These specimens were then dehydrated as above and embedded in Epon. Semithin and thin sections were cut on an LKB Ultrotome. The thin sections were collected on copper grids and contrasted with lead citrate or with uranyl acetate followed by lead citrate. For demonstration of periodate reactive substances at the ultrastructural level, tumour specimens were (d) fixed for 4 hrs in 2 • 5 per cent glutaraldehyde (o-i M phosphate buffer, pH 7-4). Following a two hour buffer rinse, the tissues were dehydrated in ethanol and styrene and embedded in Vestopal W. Thin sections were collected on gold grids, and treated with the periodic acid-silver (PA-CrA-silver) technique of Rambourg et al. (1969). All thin sections were examined in a Philips EM 300 electron microscope. Results
In semithin toluidine blue stained Epon sections, it is seen in the light microscope that cellular differentiation varies within different tumour areas. In certain regions the cells are pyramidal in shape and form acinarlike structures with central lumina. In these tumour cells there is present 948
Some ultrastructural features of acinic cell carcinoma a barely discernible population of discrete cytoplasmic granules, predominantly located in the apical portion of the cells. Compared to acinar cells of normal parotid tissue, the granules of the tumour cells are very inconspicuous at the light microscopical level (compare Figs, i and 2). In other tumour regions the cells form solid cell strands and are virtually agranular. In tumour specimens fixed in glutaraldehyde-OsO4 according to (a) and studied in the electron microscope a number of the granulated tumour cells display a fairly electron-lucid cytoplasm (light cells). However, cells with a more dense appearance (dark cells) are also observed. The latter are less common than the light cells and transitions from one type to the other are also encountered. Electron microscopy further reveals that the cytoplasmic granules of individual tumour cells differ markedly with respect to size, internal electron density and packing. In some cells, the membrane-bounded apical granules are loosely dispersed; they vary in size and display an electron dense, homogeneous appearance. In others, the granules are densely packed, are larger and more uniform in size and their contents consist of a finely flocculent material of rather low electron density. In numerous such granules a bipartite pattern is discerned, i.e. they exhibit a minute, central, dense core which is surrounded by a fairly broad rim of electron lucid material (Fig. 3).
FIG. I.
Semithin Epon section of normal parotid tissue, stained with toluidine blue. Numerous acinar cells are depicted, filled with large well-defined secretory granules. Light micrograph. X570
949
G. D. Bloom, B. Carlsoo and R. Henriksson
FIG. 2. Semithin Epon section of acinic cell carcinoma. In certain areas the tumour cells are arranged around lumen-like spaces (arrows), and in their supranuclear regions the cells display minute cytoplasmic granules. Light micrograph. X570
FIG. 3. Apical portions of two granulated tumour cells fixed in glutaraldehyde and postfixed in OsO«. A number of granules display a bipartite structure with a small dense core and a more lucid outer rim. Electron micrograph. x6-8oo
950
Some ultrastructural features of acinic cell carcinoma In specimens fixed in Millonig's fixative (b) tumour cell cytoplasm in general appears more homogeneous with respect to overall electron density. On the other hand, this technique shows up the cytoplasmic granules so electron lucid as to give the impression of membrane-bounded vesicles (Fig. 4). Also after fixation in KMnO4, (c), followed by en bloc staining with uranyl acetate and contrasting of the thin sections with lead citrate, the granules showed a similar 'empty' appearance. Nuclear polymorphism is not pronounced; the nuclei are generally rounded or oval in shape. They consistantly exhibit one or two conspicuous nucleoli and display numerous, rather regularly spaced, aggregates of chromatin at the nuclear margin. The cells in sections contain only few profiles of rough endoplasmic reticulum (RER), and only occasionally are the latter arranged in a regular pattern. Lysosome-like structures as well as lipid droplets are present in most cells but are rather inconspicuous. Golgi complexes, sometimes well-developed, appear in close proximity to the cell nucleus. The most striking feature of the tumour cell is the weird appearance of their mitochondria. Not only is their size increased but there is also a pronounced disorientation of their cristae (Fig. 5). The latter, instead of traversing the internal matrix of this organelle, often run parallel to the circumference thereby forming circles or whorl-like formations. In addition, portions of the internal membranes are vesiculated, thus creating a
FIG. 4. Granulated cell of acinic cell carcinoma specimen primarily fixed in OsO4-. All apical granules display an empty appearance. Electron micrograph. X4 75O
951
G. D. Bloom, B. Carlsoo and R. Henriksson picture resembling a string of empty beads. In some mitochondria, lastly, the internal compartment is completely occupied by microtubular structures (Fig. 5). Another characteristic feature of the cells in the present tumour is the smooth cell surfaces between adjacent cells. Lateral interdigitations are rare. Also the luminal cell surfaces are mostly even and only few microvilli are observed. In various regions along apposed cell borders subplasmalemmal bands of electron dense material are present in the cytoplasm of adjacent ells (Fig. 6). Furthermore, numerous intercellular attachments of desmosomal type may be seen. Applying the PA-CrA-silver technique to ultrathin sections of Vestopal embedded material, reaction products are observed over the cytoplasmic granules (Fig. 7). In addition, basement membrane structures stain well as do collagen fibres of the connective tissue. Finally, in certain tumour areas neoplastic cells are smaller in size and totally lack cytoplasmic granules (Fig. 8). These cells are rich in organelles and mitochondria especially are numerous. As in the granulated tumour cells mitochondrial structure is markedly altered. The cells also contain numerous cytoplasmic vacuoles the contents of which are considerably less electron dense than the cytoplasmic ground substance. Desmosomal structures are numerous and the subplasmalemmal bands of
FIG. 5. Basal and perinuclear region of tumour cell specimen fixed in OsO4. Numerous mitochondria are depicted which exhibit marked alterations in size and internal structure. Electron micrograph, x 11 • 900
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Some ultrastructural features of acinic cell carcinoma
FIG. 6. Junction area between four tumour cells. Specimen fixed in OsO«. Numerous desmosomes are observed as is a long, dense subplasmalemmal band in two apposed cells. Electron micrograph, x 19-650
FIG. 7. Glutaraldehyde fixed specimen of acinic cell carcinoma, embedded in Vestopal and stained according to the Rambourg technique. No counterstain. Numerous granules in two tumour cells exhibit a strong positive reaction. Electron micrograph. X9"3oo
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G. D. Bloom, B. Carlsoo and R. Henriksson
FIG. 8. Electron micrograph of OsO4-fixed specimen from a tumour area consisting of agranulated cells. Note the smooth cell borders as well as the dense cytoplasmic bands in the contact region of two adjacent cells (arrow). Lipid droplets and lysosomal structures are also present.
X 3•800
dense material described above are also common in these cells. No cells resembling myoepithelial or striated duct cells were encountered in the present study. 954
Some ultrastructural features of acinic cell carcinoma Discussion Persistent facial nerve paralysis, caused by a malignant parotid tumour, is a serious prognostic sign (Eneroth, 1972). The incidence of facial nerve paralysis in the acinic cell carcinoma group of malignant salivary gland tumours is, however, reported to be low. Eneroth (1972), in a series of 378 patients with malignant parotid neoplasms, recorded 46 patients with spontaneous facial paralysis. Only one of these patients had an acinic cell carcinoma. In a series of 279 malignant parotid tumours recently reviewed by Conley and Hamaker (1975), 34 patients presented involvement of the facial nerve. None of the patients with acinic cell carcinoma (a total of 17) exhibited engagement of the facial nerve. The main electron microscopic findings recorded in the present tumour are in general agreement with previous studies (Kleinsasser et al., 1967; Erlandson and Tandler, 1972; Fechner et al., 1972; Kay and Schatzki, 1972). Thus the size as well as the internal density of the cytoplasmic granules varies considerably in these tumours. However, Fechner et al. (1972) described smooth cell borders without desmosomal attachments, whereas Hiibner et al. (1968) reported an abundance of desmosomes as well as peculiar, dense, band-like structures in the most peripheral cytoplasm of the cells. The latter authors also observed well-developed intercellular interdigitations between the tumour cells. The tumour cells studied in the present investigation exhibited both smooth cell surfaces and numerous desmosome-like structures as well as the subplasmalemmal material of Hiibner et al. (1968). The mitochondrial structure of previously described cells in this type of carcinoma has not been reported as abnormal. In the present case, however, gross changes were observed in this organelle. Mitochondria of abnormal appearance are otherwise a highly characteristic feature of another salivary gland tumour, i.e. the Warthin tumour (e.g. Tandler, 1966). It should also be pointed out that structural alterations of this organelle are very common in various neoplasias (Bernhard, 1969). The zymogen granules of serous acinar cells of the major human salivary glands often exhibit, at the electron microscopic level, substructures of a characteristic appearance. Thus, in the parotid and submandibular gland the granules display a bipartite structure with a relatively dense core surrounded by a more electron-lucid outer rim (Tandler and Erlandson, 1972; Riva and Riva-Testa, 1973; Riva et al., 1974). Moreover pleomorphic secretory granules have been depicted in the salivary glands of numerous mammalian species, as well as in other exocrine cell systems (for reference see Tandler and MacCallum, 1972; Borghese et al., 1974; Bloom et al., ig77). The significance of this ordered arrangement of material within salivary gland granules is unclear. As in normal parotid tissue, the granules of numerous neoplastic cells in the described acinic cell carcinoma displayed a bipartite structure after fixation in glutaraldehyde followed by 955
G. D. Bloom, B. Carlsoo and R. Henriksson osmium tetroxide. Millonig's fixative (i per cent osmium tetroxide) as employed in the present study, gave an overall satisfactory preservation of the tissue. However, the granules of the tumour cells appeared leachedout. Similar observations have been reported in a previous study on such a tumour (Erlandson and Tandler, 1972) as well as in the normal human submandibular gland (Tandler and Erlandson, 1972). Amsterdam and Schramm (1966) found that isolated rat parotid zymogen granules, exposed to osmium, release up to 75 per cent of their protein content into the fixing fluid, resulting in a collapse of the granules. According to the same authors the granules are not affected by glutaraldehyde and retain all of their protein in this fixative. It may thus be assumed that there is a marked protein leakage from the granules of the acinic tumour cells when fixed in osmium tetroxide. From their histochemical and ultrastructural features it seems reasonable to assume that the granulated cells of acinic cell carcinoma are derived from the serous acinar cells of the salivary gland secretory units. However, the origin of the small agranular cells observed in the present report is uncertain. The intercalated duct cells of human salivary glands display many submicroscopic features in common with these agranular tumour cells. The intercalated duct cells are cuboidal in shape, exhibit smooth cell borders with numerous desmosomes, a finely filamentous cytoplasm and a poorly developed rough endoplasmic reticulum (Chisholm et al. 1974; Riva et al. 1976). It should be pointed out that Abrams and co-workers (1965) in their material (77 cases) recorded 5 acinic cell carcinomas in which the main cellular constituent was of the intercalated duct cell type. In addition Hirtzler et al. (1969) described tumour cells of two acinic cell carcinomas which at the ultrastructural level correspond to intercalated duct cells. The so-called clear cell acinic cell carcinoma is proposed to originate from striated duct cells (Echevarria, 1967). No cells whatsoever with features of striated duct cells were observed in the present study. The patient reported on in the present study exhibited a total denervation of the left facial nerve. Thus, the investigated tumour showed a local, aggressive, growth pattern. Although the overall histological picture as well as the ultrastructural appearance of the cells in general is similar to that described for acinic cell carcinomas with a more benign course, a striking difference was noted with respect to the gross pathological alterations of mitochondrial fine structure. Summary
The ultrastructure of an acinic cell carcinoma, occurring in the left parotid gland of a 52-year-old woman and causing a total facial nerve paralysis, is described. Histologically the tumour consisted of numerous granulated cells arranged around lumen-like openings and resembling a 956
Some ultrastructural features of acinic cell carcinoma secretory system. Furthermore, areas with agranulated cells growing in a solid pattern were also encountered. In the electron microscope the cytoplasmic granules of the tumour cells displayed a varied appearance. Granules of a dense homogeneous type, as well as granules with a more electron lucid appearance were observed. Furthermore, numerous cytoplasmic granules displayed a bipartite structure with a dense central and a more electron lucid outer zone. In specimens primarily fixed in OsO4 or KMnO4 the granules displayed a 'leached out' appearance. The membrane-bounded granules of the tumour cells also showed a strong positive staining with the periodic acid-chromic silver technique of Rambourg et al. (1969). Other characteristic ultrastructural features of the tumour cells studied were: Smooth cell surfaces, the presence of subplasmalemmal bands of electron dense material, desmosome-like attachment areas between cells and grossly altered mitochondria. REFERENCES ABRAMS, A. M., CORNYN, J., SCOFIELD, H. H. and HANSEN, L. S. (1965) Cancer 18,
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G. D. Bloom, B. Carlsoo and R. Henriksson RICHARDSON, K. C. (1966) Nature 210, 756. RIVA, A. and RIVA-TESTA, F. (1973) The Anatomical Record 176,149. •—, MOTTA, G. and RIVA-TESTA, F. (1974) The American Journal of Anatomy 139, 293—, TESTA-RIVA, F., DEL FIACCO, M. and LANTINI, M. S. (1976) Journal of Anatomy 122, 627. SMITH, T. E. and FECHNER, R. E. (1974) Archives of Otolaryngology 100, 324. TANDLER, B. (1966) Archives of Otolaryngology 84, 68. —, and ERLANDSON, R. A. (1972) American Journal of Anatomy 135, 419. —, and MACCALLUM, D. K. (1972) Journal of Ultrastructural Research 39, 186. Departments of Histology and Otolaryngology, University of Umea, s-901 87 Umea, Sweden.
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