Primary cutaneous adenoid cystic carcinoma: ultrastructural study and immunolocalization of Types I, III, IV, V collagens and laminin A primary adenoid cystic carcinoma ofthe skin is reported. Light microscopy revealed pseudocysts. PAS-positive basement membrane and true glandular lumen, which in aggregates are specific for adenoid cystic carcinoma. Perineural invasion was also observed. Ultrastructural examinations revealed three types of cystic spaces; pseudocysts, true glandular lumens and intercellular spaces. Enzyme histochemical examinations showed positive reactions for eccrine enzymes, including phosphorylase and succinic dehydrogenase and negative for apocrine enzymes. Immunolocalization of collagens and laminin revealed that basement membranes of the pseudocysts involve Type V collagen as well as Type IV collagen and laminin. Fukai K, Ishii M, Kobayashi H, Chanoki M, Furukawa M, Nakagawa K, Hamada T, Abe Y, Ooshima A. Primary cutaneous adenoid cystic carcinoma: ultrastructural study and immunolocalization of Types I, III, IV, V collagens and laminin. J Cutan Pathol 1990: 17: 374-380.
Adenoid cystic carcinoma (ACC) occurs most commonly in the major and minor salivary glands (1). This type of tumor has also been reported in other glandular tissues, including the tracheobronchial tree, breast, uterine cervix, larynx, esophagus, Bartholin glands ofthe vulva and prostate gland (2,3). Primary adenoid cystic carcinoma of the skin, excluding lesions ofthe external auditory canal, is very rare (4—8). Here, we report on a patient with a neoplasm with typical morphological features of ACC, and describe the uitrastructural findings and characterize the collagen types of the tumor.
Case report A 74-year-old women had a very slow growing tumor on her left buttock, for 20 years. Recently, it had become slightly tender and painful. Physical examination revealed that the lesion was a dark red, elevated, elastic hard, unmovable, non-ulcerated tumor, 3 cm in diameter (Fig. 1). No other cutaneous tumor was recognized. A biopsy indicated adenoid cystic carcinoma of the eccrine gland. The tumor
374
K. Fukai,^ M. Ishii,^ H. Kobayashi,^ M. Chanoki,^ M. Furukawa,^ K. Nakagawa,^ T. Hamada,^ Y.Ahe^ A. Ooshima^ ^Department of Dermatology Osaka City University Medical School, ^Division of Dermatology, Izumiohtsu Municipal hospital, Osaka, ^Ist Department of Pathology, Wakayama Medical College, Japan
Kazuyoshi Fukai, Department of Dermatology, Osaka City University Medical School, Osaka, Japan Accepted April 5, 1990
was excised with 1 cm margins of normal appearing skin. The surgical specimen showed a gray-yellow mass, 1 cm thick, expanding the dermis, but subcutaneous fat appeared intact. The patient remains alive without evidence of metastasis or local recurrence three years after resection.
Material and methods The specimen was fixed in a neutral buffered 10% formaldehyde solution and embedded in paraffin. Sections were stained with hematoxylin-eosin, PAS with or without diastase digestion and alcian blue. For enzyme histochemical examination, a part of the specimen was snap frozen in liquid nitrogen and 4 |im frozen sections were cut in a cryostat at — i5°C. Histochemical demonstrations were done according to standard controlled methods for succinic dehydrogenase (9), glycogen phosphorylase (10), acid phosphatase (11) and (3-glucoronidase (12). For electron microscopic examination, materials were first immersed in phosphate buffered saline for
Dormancy cutaneous ACC
cystic spaces, giving them the appearance of 'Swiss cheese' (Fig. 2). Besides this cribriform pattern of tumor nests, there were also tumor nests of strands and tubular patterns especially in the peripheral part ofthe tumor (Fig. 3). The tumor cells extended into the subcutaneous fat and perineural spaces (Fig. 4). The neopiastic cells were monomorphic and basaloid and had oval nuclei. Mitotic figures were rare. Based on histochemicai stains, two types of mucin were recognized in the specimen. One was a material within the cystic spaces that stained with PAS and which was resistant to diastase treatment; but it was not stained with alcian blue at pH 2.5. The other mucinous material was in the cystic spaces lined by PAS positive basement membrane, stained with alcian blue at pH 2.5, but not with PAS. Enzyme histochemistry
Weak reactions for glycogen phosphorylase were observed in about a half of the tumor nests. Strong reactions for succinic dehydrogenase occurred in all tumor nests. No reactions for acid phosphatase or (3-glucuronidase were shown in the specimen.
Fi^. 1. Dark-red, elevated, cla.stic hard tumor about 3 cm in diameter on the left buttock.
2 h and then fixed in 2.5% giutaraidehyde soiution and postfixed in 2% osmium tetroxide, dehydrated and embedded in Epon 812. Thin sectons were cut on a Sorvall MT2-B ultra-microtome, stained with uranyl acetate and lead citrate and examined under a Hitachi HS-9 electron microscope. For immunolocalization of Types I, III, IV and V collagens and laminin, 4 \xm cyostat sections were examined by an indirect immunofiuorescent technique. Each type of collagen used as the antigen was extracted from human placenta. Anti-type I, IV and V collagen antibodies were raised by immunizing rabbits, and monoclonal anti-type III coiiagen antibody was obtained as described elsewhere (13). Monoclonal anti-laminin antibody was perchased from Cosmo Bio Co. Ltd. The specificities of these antigens were confirmed by enzyme-linked immunosorbent assay (ELISA) and immunoblotting technique (14—16). Results Light microcopy
The epidermis was thinned with fiattened rete ridges. At the center of the tumor, the horny layer was stratified. The most remarkable feature of the tumor was the numerous cell masses with multiple 25
Culaneous Pathology
Fig. 2. Biopsy specimen showing tumor nests with cribriform pattern (H&E, X240).
375
Fukai et al.
^7:wr-y^
discernable only with electron microscopy. They were lined by cells with low density cytoplasm on the apical side which had microvilli. The true glandular lumina contained pleomorphic cellular debris, and on occasion, tubular structures about 20 nm in diameter (Fig. 8). Intercellular spaces were especially rich in solid pattern tumor nests, were varied in size and shape, and cytoplasmic processes of surrounding cells occasionally extended into the spaces. Each tumor nest was surrounded by non-oliterated, partly reduplicated basal lamina. Between narrow interstitial spaces were capillaries, collagen fibrils and fibroblasts. Immunolocalization of collagens
Fig. 3. lumor nests with tul)ular jiattern in the peripheral region of ihc tumor (H&E, X120).
Diffiise fluorescence was present in interstitial connective tissues of the specimen when treated with anti-I and III collagen antibodies (Figs. 9a,b). Type IV collagen surrounded ail tumor nests without obliteration, in most of which multiple ring-like reactions were noted, corresponding to the basement membranes of pseudocysts (Fig. 9c). On occasion. Type IV collagen accumulated within pseudocysts which were noted as fluorescent ball-like structures. In interstitial connective tissues, fine granular reactions were present. Fluorescence of Type V collagen
Electron microscopy
Two types of neopiastic cells were recognized. Most of the tumor cells were round, oval cuboidal in shape and had low electron density cytoplasm with round nuclei. They contained scattered mitochondria, rough ER, Golgi apparatus, tonofilaments, and occasional mucous granules (Fig. 5). The second type of tumor cells were triangular in shape and located around tumor nests or adjacent to the pseudocysts and had electron dense cytoplasm. They contained rough ER and scattered mitochondria (i'ig. 5). Despite careful examination, there were no fibrillar components in these cells. This type of ceil lay in the tumor nests with cibriform patterns, but not in those with solid patterns. All these tumor cells were connected with each other by desmosomes. I^seudocysts and true glandular lumina occurred in more than half of the nests (Fig. 6). Round, ovai and occasionally polygonal pseudocysts, which corresponded to cribriform cystic spaces observed by light microscopy, were lined by a conspicuous basal lamina which occasionally reduplicated (Fig. 7). in the pseudocysts, numerous stellate granules, which were considered to be proteoglycans, were present. Although some true lumina were apparent with Epon-embcddcd thick sections, most of them were 376
r
:/
V r'
Fig. 4. Perineural invasion of t u m o r cells ( H & E , X120).
Dormancy cutaneous ACC Fig. 5. Electron micrograph of a cutaneous ACC showing two types of tumor cells: basaloid and myoepithelial cells. True glandular lumen is in the center and the tumor nest is surrounded by basement membrane (X2,7OO).
occurred in ring-like patterns in tumor nests as noted with anti-Type IV collagen antibodies, as well as in diffuse patterns in interstitial connective tissues (Fig. 9d). Staining patterns with anti-laminin andibody were almost the same as those of Type IV collagen, while fine granular reaction in interstitial connective tissue was absent (Fig. 9e).
Discussion ACC is a common neoplasm of major and minor salivary glands. According to Thackray and Lucas, it accounts for 4.4% of all tumors of the major salivary glands and 1.2% of parotid gland tumors, and it was defined as having a characteristic cribriform appearance and to comprise of two types of tumor ceils, duct-lining cells and cells of myoepitheiial type (1). The term, adenoid cystic carcinoma, has aiso been applied to neoplasms of the laerimal gland, respiratory tract, breast, and cervix, which have the same characteristic histological features
(2). Primary cutaneous adenoid cystic carcinoma is very rare. To date, about twenty cases have been reported (4—8). They were described as very slow growing, usually asymptomatic, discrete nodules affecting middle aged and older individuals. Common sites were the scalp and chest. Local recurrences occurred in half of the reported cases and metastasis occurred in two. Histologically, cutaneous adenoid cystic carcinoma is indistinguishable from extra-cutaneous adenoid cystic carcinoma. Cutaneous adenoid cystic carcinoma exhibits characteristic tumor nests with cribriform patterns as well as nests with tubular and solid patterns (4,5). Cribriform nests have two types of cystic spaces: pseudocysts and true glandular iumens. These histological features arc distinct from adenoid basal cell epithelioma (8) and microcystic adnexal carcinoma (17). Electronmicroscopic study of this case also revealed similar ultrastructural features as reported with extracutaneous ACCs (2,3, 18-24) and recently
\ Fig. 6. Pseudolumen is lined by basement membrane without obliteration, and true glandular lumen is lined by microvilli (X 8,500). 25*
377
Fukai et al. Fig. 7. Reduplicated basement membranes of pseudolumen. Tumor cells contained mucous granules (X 14,000).
reported cutaneous (ACC (7). The tumor cells were connected to each other by desmosomes and three types of cystic spaces, namely true glandular lumens, pseudocysts, and intercellular spaces, were observed. Basement membranes lined the inside of the pseudocysts and the outside of the tumor nests without obliteration, which may suggest benign biological behavior of the tumor. Some tumor cells were similar to myoepithelial cells, but we could not detect myofibrils in the cytoplasm. There arc several reports that demonstrated the involvement of myoepithelial cells in ACCs (2,25,25). Further study and accumulation of this type of tumor are necessary for the question of myoepithelial participation in the genesis of primary cutaneous ACC. An unusual finding in this study was the numerous tubular structures which were located in true lumens. There is only one report describing the
tubular structures in true glandular lumens in ACC ofthe breast (22). It is still not clear whether this structure is characteristic of this type of tumor. i"urther study is needed to elucidate the exact nature of the structures. Basement membranes in pseudocysts were, in this study, demonstrated to contain Types iV and V collagen and laminin. Our previous report revealed that the basement membrane of tiie epidermai-dermal junction in normal human skin does not involve Type V collagen (16). Ultrastructural features of basement membranes of pseudocysts were unique because of their thicker basal lamina and occasional reduplications. Participation of Type V collagen in the basement membrane may contribute to the morphological alterations. The origin of cutaneous ACC remains controversial (4,7). In this study, the presence of eccrine
Fig. 8. Higher magnidcation of the central portion of Fig. (3. True lumctis often included tube-like structures (xr^O,OOO).
378
Dormancy cutaneous ACC
Fig. 9. Imnuinolocalization of 1 yi^cs I, H I , IV a n d V collagen and l a m i n i n . Dillusc i m n i u n o n u o i c s c e n c c in interstitial connective tissues was noted when r('a( ted with Ty|)
Adenoid cystic carcinoma (ACC) occurs most commonly in the major and minor salivary glands (1). This type of tumor has also been reported in other glandular tissues, including the tracheobronchial tree, breast, uterine cervix, larynx, esophagus, Bartholin glands ofthe vulva and prostate gland (2,3). Primary adenoid cystic carcinoma of the skin, excluding lesions ofthe external auditory canal, is very rare (4—8). Here, we report on a patient with a neoplasm with typical morphological features of ACC, and describe the uitrastructural findings and characterize the collagen types of the tumor.
Case report A 74-year-old women had a very slow growing tumor on her left buttock, for 20 years. Recently, it had become slightly tender and painful. Physical examination revealed that the lesion was a dark red, elevated, elastic hard, unmovable, non-ulcerated tumor, 3 cm in diameter (Fig. 1). No other cutaneous tumor was recognized. A biopsy indicated adenoid cystic carcinoma of the eccrine gland. The tumor
374
K. Fukai,^ M. Ishii,^ H. Kobayashi,^ M. Chanoki,^ M. Furukawa,^ K. Nakagawa,^ T. Hamada,^ Y.Ahe^ A. Ooshima^ ^Department of Dermatology Osaka City University Medical School, ^Division of Dermatology, Izumiohtsu Municipal hospital, Osaka, ^Ist Department of Pathology, Wakayama Medical College, Japan
Kazuyoshi Fukai, Department of Dermatology, Osaka City University Medical School, Osaka, Japan Accepted April 5, 1990
was excised with 1 cm margins of normal appearing skin. The surgical specimen showed a gray-yellow mass, 1 cm thick, expanding the dermis, but subcutaneous fat appeared intact. The patient remains alive without evidence of metastasis or local recurrence three years after resection.
Material and methods The specimen was fixed in a neutral buffered 10% formaldehyde solution and embedded in paraffin. Sections were stained with hematoxylin-eosin, PAS with or without diastase digestion and alcian blue. For enzyme histochemical examination, a part of the specimen was snap frozen in liquid nitrogen and 4 |im frozen sections were cut in a cryostat at — i5°C. Histochemical demonstrations were done according to standard controlled methods for succinic dehydrogenase (9), glycogen phosphorylase (10), acid phosphatase (11) and (3-glucoronidase (12). For electron microscopic examination, materials were first immersed in phosphate buffered saline for
Dormancy cutaneous ACC
cystic spaces, giving them the appearance of 'Swiss cheese' (Fig. 2). Besides this cribriform pattern of tumor nests, there were also tumor nests of strands and tubular patterns especially in the peripheral part ofthe tumor (Fig. 3). The tumor cells extended into the subcutaneous fat and perineural spaces (Fig. 4). The neopiastic cells were monomorphic and basaloid and had oval nuclei. Mitotic figures were rare. Based on histochemicai stains, two types of mucin were recognized in the specimen. One was a material within the cystic spaces that stained with PAS and which was resistant to diastase treatment; but it was not stained with alcian blue at pH 2.5. The other mucinous material was in the cystic spaces lined by PAS positive basement membrane, stained with alcian blue at pH 2.5, but not with PAS. Enzyme histochemistry
Weak reactions for glycogen phosphorylase were observed in about a half of the tumor nests. Strong reactions for succinic dehydrogenase occurred in all tumor nests. No reactions for acid phosphatase or (3-glucuronidase were shown in the specimen.
Fi^. 1. Dark-red, elevated, cla.stic hard tumor about 3 cm in diameter on the left buttock.
2 h and then fixed in 2.5% giutaraidehyde soiution and postfixed in 2% osmium tetroxide, dehydrated and embedded in Epon 812. Thin sectons were cut on a Sorvall MT2-B ultra-microtome, stained with uranyl acetate and lead citrate and examined under a Hitachi HS-9 electron microscope. For immunolocalization of Types I, III, IV and V collagens and laminin, 4 \xm cyostat sections were examined by an indirect immunofiuorescent technique. Each type of collagen used as the antigen was extracted from human placenta. Anti-type I, IV and V collagen antibodies were raised by immunizing rabbits, and monoclonal anti-type III coiiagen antibody was obtained as described elsewhere (13). Monoclonal anti-laminin antibody was perchased from Cosmo Bio Co. Ltd. The specificities of these antigens were confirmed by enzyme-linked immunosorbent assay (ELISA) and immunoblotting technique (14—16). Results Light microcopy
The epidermis was thinned with fiattened rete ridges. At the center of the tumor, the horny layer was stratified. The most remarkable feature of the tumor was the numerous cell masses with multiple 25
Culaneous Pathology
Fig. 2. Biopsy specimen showing tumor nests with cribriform pattern (H&E, X240).
375
Fukai et al.
^7:wr-y^
discernable only with electron microscopy. They were lined by cells with low density cytoplasm on the apical side which had microvilli. The true glandular lumina contained pleomorphic cellular debris, and on occasion, tubular structures about 20 nm in diameter (Fig. 8). Intercellular spaces were especially rich in solid pattern tumor nests, were varied in size and shape, and cytoplasmic processes of surrounding cells occasionally extended into the spaces. Each tumor nest was surrounded by non-oliterated, partly reduplicated basal lamina. Between narrow interstitial spaces were capillaries, collagen fibrils and fibroblasts. Immunolocalization of collagens
Fig. 3. lumor nests with tul)ular jiattern in the peripheral region of ihc tumor (H&E, X120).
Diffiise fluorescence was present in interstitial connective tissues of the specimen when treated with anti-I and III collagen antibodies (Figs. 9a,b). Type IV collagen surrounded ail tumor nests without obliteration, in most of which multiple ring-like reactions were noted, corresponding to the basement membranes of pseudocysts (Fig. 9c). On occasion. Type IV collagen accumulated within pseudocysts which were noted as fluorescent ball-like structures. In interstitial connective tissues, fine granular reactions were present. Fluorescence of Type V collagen
Electron microscopy
Two types of neopiastic cells were recognized. Most of the tumor cells were round, oval cuboidal in shape and had low electron density cytoplasm with round nuclei. They contained scattered mitochondria, rough ER, Golgi apparatus, tonofilaments, and occasional mucous granules (Fig. 5). The second type of tumor cells were triangular in shape and located around tumor nests or adjacent to the pseudocysts and had electron dense cytoplasm. They contained rough ER and scattered mitochondria (i'ig. 5). Despite careful examination, there were no fibrillar components in these cells. This type of ceil lay in the tumor nests with cibriform patterns, but not in those with solid patterns. All these tumor cells were connected with each other by desmosomes. I^seudocysts and true glandular lumina occurred in more than half of the nests (Fig. 6). Round, ovai and occasionally polygonal pseudocysts, which corresponded to cribriform cystic spaces observed by light microscopy, were lined by a conspicuous basal lamina which occasionally reduplicated (Fig. 7). in the pseudocysts, numerous stellate granules, which were considered to be proteoglycans, were present. Although some true lumina were apparent with Epon-embcddcd thick sections, most of them were 376
r
:/
V r'
Fig. 4. Perineural invasion of t u m o r cells ( H & E , X120).
Dormancy cutaneous ACC Fig. 5. Electron micrograph of a cutaneous ACC showing two types of tumor cells: basaloid and myoepithelial cells. True glandular lumen is in the center and the tumor nest is surrounded by basement membrane (X2,7OO).
occurred in ring-like patterns in tumor nests as noted with anti-Type IV collagen antibodies, as well as in diffuse patterns in interstitial connective tissues (Fig. 9d). Staining patterns with anti-laminin andibody were almost the same as those of Type IV collagen, while fine granular reaction in interstitial connective tissue was absent (Fig. 9e).
Discussion ACC is a common neoplasm of major and minor salivary glands. According to Thackray and Lucas, it accounts for 4.4% of all tumors of the major salivary glands and 1.2% of parotid gland tumors, and it was defined as having a characteristic cribriform appearance and to comprise of two types of tumor ceils, duct-lining cells and cells of myoepitheiial type (1). The term, adenoid cystic carcinoma, has aiso been applied to neoplasms of the laerimal gland, respiratory tract, breast, and cervix, which have the same characteristic histological features
(2). Primary cutaneous adenoid cystic carcinoma is very rare. To date, about twenty cases have been reported (4—8). They were described as very slow growing, usually asymptomatic, discrete nodules affecting middle aged and older individuals. Common sites were the scalp and chest. Local recurrences occurred in half of the reported cases and metastasis occurred in two. Histologically, cutaneous adenoid cystic carcinoma is indistinguishable from extra-cutaneous adenoid cystic carcinoma. Cutaneous adenoid cystic carcinoma exhibits characteristic tumor nests with cribriform patterns as well as nests with tubular and solid patterns (4,5). Cribriform nests have two types of cystic spaces: pseudocysts and true glandular iumens. These histological features arc distinct from adenoid basal cell epithelioma (8) and microcystic adnexal carcinoma (17). Electronmicroscopic study of this case also revealed similar ultrastructural features as reported with extracutaneous ACCs (2,3, 18-24) and recently
\ Fig. 6. Pseudolumen is lined by basement membrane without obliteration, and true glandular lumen is lined by microvilli (X 8,500). 25*
377
Fukai et al. Fig. 7. Reduplicated basement membranes of pseudolumen. Tumor cells contained mucous granules (X 14,000).
reported cutaneous (ACC (7). The tumor cells were connected to each other by desmosomes and three types of cystic spaces, namely true glandular lumens, pseudocysts, and intercellular spaces, were observed. Basement membranes lined the inside of the pseudocysts and the outside of the tumor nests without obliteration, which may suggest benign biological behavior of the tumor. Some tumor cells were similar to myoepithelial cells, but we could not detect myofibrils in the cytoplasm. There arc several reports that demonstrated the involvement of myoepithelial cells in ACCs (2,25,25). Further study and accumulation of this type of tumor are necessary for the question of myoepithelial participation in the genesis of primary cutaneous ACC. An unusual finding in this study was the numerous tubular structures which were located in true lumens. There is only one report describing the
tubular structures in true glandular lumens in ACC ofthe breast (22). It is still not clear whether this structure is characteristic of this type of tumor. i"urther study is needed to elucidate the exact nature of the structures. Basement membranes in pseudocysts were, in this study, demonstrated to contain Types iV and V collagen and laminin. Our previous report revealed that the basement membrane of tiie epidermai-dermal junction in normal human skin does not involve Type V collagen (16). Ultrastructural features of basement membranes of pseudocysts were unique because of their thicker basal lamina and occasional reduplications. Participation of Type V collagen in the basement membrane may contribute to the morphological alterations. The origin of cutaneous ACC remains controversial (4,7). In this study, the presence of eccrine
Fig. 8. Higher magnidcation of the central portion of Fig. (3. True lumctis often included tube-like structures (xr^O,OOO).
378
Dormancy cutaneous ACC
Fig. 9. Imnuinolocalization of 1 yi^cs I, H I , IV a n d V collagen and l a m i n i n . Dillusc i m n i u n o n u o i c s c e n c c in interstitial connective tissues was noted when r('a( ted with Ty|)
