104
0 1992
The Japanese Society of Pathology
HistochemicaI Demonst rat io n of Ga st ro intest ina I M ucins in Ovarian Mucinous Cystadenoma
Tanri Shiozawa1*2,Yoshiharu Tsukahara', Keiko Ishi?, Hiroyoshi Ota2, Jun Nakayama2, and Tsutomu Katsuyama2
Forty-one specimens of mucinous cystadenoma (MCA) of the ovary and 13 specimens of normal uterine endocervix were investigated histochemically using alcian blue pH 2.5PAS, high iron diamine-alcian blue pH 2.5, galactose oxidase-Schiff reaction (GOS, for detection of gastric surface mucous cell mucins), paradoxical Concanavalin A staining (PCS, for detection of gastric gland cell mucins) and periodic acid-sodium borohydride-potassium hydroxide-PAS (PA-SB-PH-PAS) reaction (for detection of mucins of the large intestine). In addition, Grimelius staining was carried out to explore the distribution of endocrine cells. The MCAs contained abundant neutral mucins but a little acid mucins, whereas sulfomucins predominated i n normal endocervices. Among the MCAs, 30,26,2 and 9 had tumor cells positive for GOS, PCS, PASB-PH-PAS and Grimelius stain, respectively. These results indicate that MCAs frequently contain tumor cells of the gastrointestinal type and endocrine cell type. Endocervical epithelia, on the other hand, lack reactivity for PCS, PA-SB-PH-PAS and Grimelius stain, although i n 8 of the present specimens, the lining cells showed weak GOS reactivity. Acta Pathol Jpn 42 : 104-110, 1992. Key words : Ovarian mucinous cystadenoma, Uterine cervix,
Mucin, Grimelius stain, Histochemistry
Mucinous cystadenoma (MCA) is a common ovarian neoplasm. Characteristically, it contains abundant epithelial mucins, and the tumor cells lining the cyst produce mucins. There has been considerable debate over the histogenesis of this tumor, and early reports referred to the teratoma (1-4) and metaplasia (5-9)
Received May 23, 1991. Accepted for publication October 14, 1991. Departments of 'Gynecology and Obstetrics, and 'Laboratory Medicine, Shinshu University School of Medicine, Matsumoto. Mailing address: Tsutomu Katsuyama, M.D., Ph.D., Department of Laboratory Medicine, Shinshu University School of Medicine, Asahi 3-1-1, Matsumoto 390, Japan.
theories. In this study, we attempted to characterize the tumor cells of MCA using histochemical techniques for differentiation of mucins. In addition, uterine endocervical mucous cells were examined by the same techniques in order to compare their histochemical properties with those of the tumor cells. The results indicated that most MCAs contained mucous cells characteristic of the gastrointestinal mucosa. The mucins in MCAs were found to differ from those in endocervical cells, which lacked the properties characteristic of gastrointestinal m uci ns.
MATERIALS AND METHODS Resected tumor specimens were obtained from 41 patients with MCA of the ovary. The patients ranged in age from 11 to 81 yr, with a mean of 42.3yr. In addition, 13 uterine cervices were examined. These were obtained from uteri removed because of leiomyoma, and were histologically normal. They included 8 specimens in the proliferative, 3 in the secretory and 2 in the postmenopausal phases. All tissues were fixed in buffered 10% formalin and em bedded in paraffin. T hree-micro meter-t hic k seria I sections were prepared and stained with hematoxylin and eosin (HE). In addition, they were stained with alcian blue pH 2.5-PAS (AB-PAS) (lo), high iron diamine-alcian blue pH 2.5 (HID-AB) (1l), galactose oxidaseSchiff (GOS) (12), paradoxical Concanavalin A staining (PCS) (13) and periodic acid-sodium borohydride-potassium hydroxide-periodic acid-Schiff (PA-SB-PH-PAS) (14). Adiacent tissue sections were also stained by the Grimelius technique. The histochemical specificities of these methods and the sources of the reagents are listed in Table 1.
Acta Pathologica Japonica 42 (2) : 1 9 9 2
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Table 1. Histochemical Methods Employed and Their Specificities
Method (abbreviation) Alcian blue pH 2.5-PAS (A B- PAS) High iron djamine-alcian blue pH 2.5 (HI D-AB) Galactose oxidase-Schiff (GOS) Paradoxical Concanavalin A staining (PCS)a Periodic acid-sodium borohydride-potassium hydroxide-PAS (PA-SB-PH-PAS) Grimelius stain
Specificity Detects mucins and differentiates acid and neutral mucins Differentiates sialo- and sulfomucins Identifies mucins of gastric surface mucous cells Identifies mucins of gastric gland mucous cells Identifies sialic acid with 0-acetyl group(s) on C,-,-,, characteristic for goblet cells of large intestine Identifies endocrine cells
Reagent m -dia mineb p-diarninec Galactose oxidasec Concanavalin Ac
This table is modified from that in the report by Akamatsu and Katsuyama (17). a By PCS, epithelial mucins are classified into three groups. In this study, only the sequence for identifying class Ill rnucins, which is a hallmark of gastric gland mucous cells, was carried out. b Kanto Chemicals, Tokyo, Japan. C Sigma, St. Louis, Missouri, USA.
RESULTS Histology The 41 cases of MCA included 34 benign cases and 7 cases containing lesions of borderline malignancy (Table 2). The tumor cells were mostly columnar cells containing abundant mucins in their apical cytoplasm (Fig. la). Cuboidal or columnar cells with faintly eosinophilic cytoplasm, which contained little mucosubstances, and goblet cell-type tumor cells were seen less frequently (Fig. 1b). Endocrine cells and Paneth cells were not clearly evident by HE staining. The first type corresponded to the so-called endocervical cells described by Fenoglio et a/. (8). Their “intestinal-type’’ epithelial cells consisted of goblet cells and argyrophil cells intermingled with “absorptive cells’’, compatible with the latter cell group in our study. Fenoglio et a/. (8, 9) classified MCA into three groups. When the lining epithelium was composed of a monotonous arrangement of mucin-filled columnar cells with basal nuclei, MCA was classified as the endocervical type. On the other hand, when the lining epithelium consisted of absorptive cells, goblet cells and argyrophil cells, the MCA was classified as the intestinal type. MCA was classified as being of mixed type if the tumor contained both intestinal- and endocervical-type cells. Table 2. Classification of Ovarian Mucous Cystadenomas Examined
Benign Borderline malignancy
Endocervical tvoe Mixed tvPe 23 11 3 4
Tumors showing borderline malignancy were also classified following these criteria (Figs. l c , d). Twentysix of the cases examined in the present study were of the endocervical type, and 15 were of the mixed type. None were classified as belonging to the intestinal type. Three cases also had a dermoid cyst, but Brenner nests were not found in any of them. In about half of the cases examined, the tumor cells occasionally formed gland-like structures (Fig. le). They had clearer cytoplasm than ordinary endocervicaltype tumor cells, and in adjacent sections, these cells occasionally stained for class Ill mucins. In normal uterine cervices, the lining mucous cells resembled mucous cell-type tumor cells, but had a smaller amount of cytoplasm (Fig. lf). They did not show significant morphological changes between the two menstrual phases, or between the reproductive and menopausal states. Histochemistry Results obtained by histochemical staining are summarized in Table 3.
AB-PAS : In MCAs, endocervical-type tumor cells exhibited intense PAS reactivity, but mostly showed absence of, or only faint alcianophilia (Fig. 2a). In cases showing borderline malignancy, intracytoplasmic mucins tended to be reduced, and cell coat-type mucins, which present on the apical surface, were more evident (Fig. 2b). However, the acidity of mucins was consistent with ordinary endocervical-type tumor cells. Goblet cell-type tumor cells revealed intense alcianophilia, and “absorptive cells” possessed surface coats (Fig. 2c). In the normal endocervices, mucous cells lining either
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Mucins of Ovarian Mucinous Cystadenoma (Shiozawa et a/.)
Figure 1. Histology of mucinous cystadenoma (MCA). HE. a : Endocervical-type mucinous cystadenoma (MCA). Note orderly array of tall columnar cells with basal nuclei. b : Mixed-type MCA. Goblet cells (arrows) are intermingled with “absorptive cells”. c : Endocervical type with borderline malignancy. Slight cellular atypia is evident. d : Mixed type with borderline malignancy. Note slight nuclear atypia. Arrows indicate goblet cell-type tumor cells. e : Endocervical type. Gland-like indented portions are evident. cf. Figure 3a. f : Normal endocervical mucous cells. The cytoplasm is generally smaller in extent than that of endocervical-type tumor cells.
the cervical surface or clefts showed intense staining for acid mucins (Fig. 2d). The acidity was consistent, irrespective of sexual phase.
HID-AB : In MCAs, acid mucins were detected in 38 cases. A l l specimens had sialomucins, which predominated in 35 cases. Alcianophilia of endocentical-
type tumor cells was, however, much less marked than that of endocervical lining cells (Fig. 2e), and occasionally no staining was seen. Twenty cases had sulfomucins, which predominated in only 3 cases. All 3 of the cases which lacked acid mucins were of the benign endocervical type. Goblet cell-type tumor cells consistently showed alcianophilia, indicating the presence of
Figure 2. Histochemical reactivities of MCAs and endocervical epithelia. Figures 2a to 2 d were prepared from tissue sections stained with AB pH 2.5-PAS, Figures 2e t o 2g from tissue sections stained with HID-AB pH 2.5 and Figures 2h t o 2 j from tissue sections stained with GOS reaction. a : Endocervical-type MCA. Tumor cells reveal intense PAS reactivity. b : Endocervicaltype MCA with borderline malignancy. lntracytoplasmic mucins are almost absent. Counterstained with hematoxylin. c : Mixed-type MCA. Goblet cells show intense alcianophilia. Note the surface coat on the absorptive cells. d : Normal endocervical mucous cells. These cells show intense alcianophilia. e : Endocervical-type MCA. Only faint or negligible alcianophilia IS evident. f : Mixed-type MCA. Goblet cells reveal intense alcianophilia. g : Normal endocervical mucous cells. Marked HID reactivity is noted. h : Endocervical-type MCA. Tall columnar cells show intense reactivity. i : Mixed-type MCA. Goblet cells (arrows) lack reactivity. j : Normal endocervical mucous cells. These cells are also GOS-reactive, but stain only faintly. Counterstained with hematoxylin.
Acta Pathologica Japonica 4 2 (2) : 1992
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Table 3. Histochemical Reactivities of Mucous Cells in Ovarian Mucous Cystadenoma
AB-PASa R>B R=B Rsulfo sialo = sulfo sialo 50% 50-10% B means that PAS reactivity of tumor cells predominates compared with their alcianophilia, but includes cases which lack alcianophilia. R < B means that alcianophilia of tumor cells predominates and includes those in which all tumor cells possess alcianophilia. b sialo>sulfo means that sialomucin is more abundant than sulfomucin, but includes cases which lack sulfomucin. There were no cases which contained sulfomucin only.
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Mucins of Ovarian Mucinous Cystadenoma (Shiozawa et a/.)
Figure 3. Other histochemical reactivities of MCA. a : Endocervical-type MCA. Prepared from the section adjacent to that used for Figure 2. Basal regions (arrows) of gland-like portions stain for class Ill mucins. PCS. b : Mixed-type MCA. Goblet cells contain 8-0-acetylated sialic acid. PA-SB-PH-PAS. c : Mixed-type MCA. Endocrine cells (arrows) are intermingled with “absorptive cells”. Grimelius stain.
sialomucins (Fig. 2f). On the other hand, sulfomucins were distinctly dominant in the endocervical lining cells (Fig. 2g). GOS: In 30cases GOS-reactive tumor cells were demonstrated. Columnar cells showing intense PAS reactivity were stained distinctly with this method (Fig. 2h). In 18 of 30 cases, 10-50% of tumor cells showed GOS reactivity. An incidence of over 50% was found in only 2 cases. No correlation was noted between atypia and GOS reactivity. Goblet cells lacked GOS reactivity almost entirely (Fig. 2i). Among 13 normal endocervical specimens, 8 were positive for the GOS reaction, although the intensity of staining was much lower than that of GOS-reactive tumor cells of MCAs (Fig. 2j).
PCS : Columnar cells containing class Ill mucins were demonstrated in 2 6 cases, which included 1 7 cases of the endocervical type and 9 of the mixed type. The positive cells usually occurred in the indented regions of gland-like portions, although they were also found among the ordinary endocervical-type tumor cells (Fig. 3a). Their incidence in each case was only a few percent at maximum. Compared with adjacent sections stained with AB-PAS or HID-AB, positive cells were mostly stained for neutral or weakly acidic mucins. Strongly acidic cells never revealed class Ill mucins. Among 2 6
positive cases, 2 2 also contained GOS-reactive cells. Class Ill mucin-containing cells showed weak or negligible GOS reactivity, and vice versa. Tumor cells showing borderline malignancy lacked class Ill reactivity. In normal endocervices, no lining cells showed class Ill reactivity. PA-SB-PH-PAS : Among 41 cases, only 2 contained tumor cells showing reactivity for 8-0-acetylated sialic acid. All of these were of the goblet cell type (Fig. 3b). In normal endocervices, no lining cells stained for 8-0acetylated sialic acid. Grimelius stain : Argyrophil cells were demonstrated in 9 cases (Fig. 3c). There was no relation between the distribution of argyrophil cells and other types of tumor cell. No endocervical epithelia showed this reaction.
DISCUSSION One of the most striking features of MCA is its ability to synthesize and secrete mucins, a property that gives the cystic fluid its characteristic mucinous appearance. Characterization of these mucins would provide information to help clarify the histogenesis of this tumor. Tumor cells responsible for retention of mucins can be classified into two types; the endocervical type and goblet cell type. The former type is the predominant
Acta Pathologica Japonica 42 (2) : 1992
one, resembling mucous cells lining the uterine endocervix in possessing high columnar cytoplasm filled with mucins. The latter resembles the goblet cells present in the gastrointestinal tract, as well as those in the respiratory tract. Histochemical studies on ”endocervicaltype” tumor cells, however, indicated that the tumor cells frequently contained gastric-type mucins. Thus, with regard to mucin acidity, endocervical-type tumor cells resembled gastric surface mucous cells. In normal human tissues, covering epithelia consisting exclusively of columnar mucous cells are found only in the stomach and endocervix. The two epithelia can hardly be distinguished in ordinary preparations stained with HE. Gastric surface mucous cells, however, are characterized by a content of neutral mucins almost exclusively (15), whereas other covering epithelia of the mucous cell type consistently stain for either sialomucins or sulfomucins, as exemplified by goblet cells of the respiratory and gastrointestinal tracts, and columnar epithelia lining the pancreatic ducts and biliary tract. As confirmed in this study, endocervical mucous cells also contained highly acidic mucins, and therefore differed from “endocervical-type” tumor cells. The present study also demonstrated that some of the endocervical-type cells contained class Ill mucins. These cells were usually located in the indented portions, or lined gland-like structures in the stroma. In all vertebrates examined previously, class Ill mucins, which are specifically demonstrated by PCS, have been found only in the cardiac glands, mucous neck cells, pyloric glands and Brunner‘s glands (10, 15, 16). Class Ill Con A reactivity is a useful marker for identifying these cells, and its presence indicates that the cells containing these mucins have properties similar t o those of the glandular epithelia described above. GOS reactivity of the endocervical-type tumor cells resembled that of gastric surface mucous cells. In the gastrointestinal and respiratory tracts, gastric surface mucous cells show the most intense GOS reactivity (10, 15, 17). Endocervical-type tumor cells also showed relatively intense GOS reactivity, whereas the endocervical mucous cells stained rather faintly. It is noteworthy that in both MCAs and the stomach, mucous cells showing intense GOS reactivity lack class Ill Con A reactivity (18). However, GOS-reactive tumor cells were demonstrated in only 30 of 41 cases. In the human stomach, the surface mucous cells, which exhibit intense blood group activity, may show a rather weak GOS reaction(l8). This phenomenon could be due to the fact that galactose oxidase selectively modifies the p-anomer of galactose and N-acetylgalactosamine, whereas the a-anomer of these two sugar residues occupies the non-reducing terminal of blood group B and
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A determinants, respectively. Although blood group activity of tumor cells in MCAs was not examined in this study, endocervical-type tumor cells which showed weak GOS reactivity might contain mucins with blood group activity. The present observations indicated that so-called endocervical-type tumor cells included gastric-type mucous cells, but did not exclude the possible existence of real endocervical-type tumor cells in MCAs. A specific marker for the latter cell type is a prerequisite for demonstration of its presence in MCAs. 8-0-Acetylated sialic acid was demonstrated in two cases. Among various human organs, 8-0-acetylated sialic acid is most abundant in goblet cells of the large intestine (14), and the PA-SB-PH-PAS sequence has been employed to detect metastatic bowel carcinoma (19). Lapertosa et a/. (20) found this sialic acid in goblet cell-type tumor cells of MCAs in over 50% of cases examined. The difference in the incidence of positive goblet cells between our study and that of Lapertosa et a/. might reflect the number of blocks examined in each case. Since most goblet cell-type tumor cells contained ordinary sialic acid, these cells might possess properties of small-intestinal goblet cells. By immunizing rabbits with high-molecular-weight proteins (HMWP) purified from gastric mucosa and the contents of MCAs, Bara et a/. (21) obtained antisera against gastric HMWP and MCA HMWP, respectively. Both of the antisera reacted with both gastric mucosa and MCA, indicating the presence of macromolecules bearing epitopes common to these two tissues. Their results also suggested the presence of antigens common to the gastric mucosa, colon mucosa and MCA, and agree well with our findings. Bara et a/., however, did not refer to the location of the epitopes responsible for these reactions, i.e. on the peptide portions or saccharide portions of the macromolecules purified. As confirmed in the present study, endocrine cells have been noted in MCA. Recent studies have shown that APUD cells occur more widely in human tissues than believed previously. Although we could not identify argyrophil cells in the endocervix, Fox et a/. (22) and Satake et a/. (23) noted their presence a t the same site. Moreover, Satake and Matsuyama (24) found endocrine cells in the endometrium and noted that they contained serotonin and somatostatin. The demonstration of endocrine cells, therefore, would not be a hallmark of endoderma I a nd gas t rointest ina I epithelia I origin. Several theories have been proposed for the histogenesis of MCA, including the teratoma (1-4) and metaplasia (5-8) theories. The latter asserts that all types of tumor cell are derived from metaplasia of the coelomic epithelium, whereas, in the former, the mucous
Mucins of Ovarian Mucinous Cystadenoma (Shiozawa et a/.)
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cells of MCA are explained as being due t o clonal proliferation of a particular cell species, a s exemplified by struma ovarii. However, neither intestinal metaplasia nor gastric metaplasia has been observed in the mesothelium or the female genital tract. Moreover, tumor cells showing a gastric phenotype have never been found in carcinomas of the female genital tract except for cystadenocarcinoma of the ovary (Shiozawa and Katsuyama, unpublished observation). In teratoma, o n the other hand, gastrointestinal epithelia are frequently a major constituent. Thus the teratoma theory seems better fitted t o our present results.
12.
13.
14.
15.
REFERENCES 1. Meyer R.
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Zur Histogenese und Einteilung der Ovarialkystome. Ein Kritische Litratursichtung. Monatschr Geburtschilfe Gynaecol 44: 302-358, 1916. Masson P. Sur la prssence des cellules argentaffines dans les cystes pseudomucineux de I'ovarie. Union Med Can 6 7 : 2-5, 1938. Beck RP and Latour JPA. Review of 1019 benign ovarian neoplasm. Obstet Gynecol 16 : 482-497, 1960. Langley FA, Cummins PA, and Fox H. An ultrastructural study of mucin secreting epithelia in ovarian neoplasm. Acta Pathol Microbiol Scand [A] 80 (SUPPI233) 76-78, 1972. Towers RP. A note on the origin o f the pseudomucinous cystadenoma of the ovary. J Obstet Gynaecol Br Emp 6 3 : 253-258, 1956. Evans RW, Harris HR, and McDougall CDM. Argentaffin carcinoma (carcinoid tumor) of ovary. J Clin Pathol 1 2 : 183-187, 1959. Azzopardi JG and Tsun HL. Intestinal metaplasia with argentaffin cells in cervical adenocarcinoma. J Pathol 90 : 686-690, 1965. Fenoglio CM, Ferenczy A, and Richart RM. Mucinous tumor of the ovary. Ultrastructural studies of mucinous cystadenomas with histogenetic considerations. Cancer 36 : 1709-1 722, 1975. Fenoglio CM, Cottral GA, Ferenczy A, et a/. Mucinous tumors of the ovary. Ill. Histochemical studies. Gynecol Oncol 4 : 151-157, 1976. Spicer SS, Horn RG, and Leppi TJ. Mucosubstance histochemistry of the connective tissue. ln Wagner BM and Smith DE, eds. The Connective Tissue. Williams & Wilkins, Baltimore, 1967 : 251 -303. Spicer SS. Diamine methods for differentiating
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mucosubstances histochemically. J Histochem Cytochem 1 3 : 211-234, 1965. Katsuyama T, Ono K, Nakayama J, and Kanai M. Recent advances in mucosubstance histochemistry. In Kawai K, ed. Gastric Mucus and Mucus Secreting Cells. Excerpta Medica, Amsterdam, 1985 : 3-18, Katsuyama T and Spicer SS. Histochemical differentiation of complex carbohydrates with variants of the Concanavalin A-horseradish peroxidase method. J Histochem Cytochem 2 6 : 233-250, 1978. Culling CFA, Reid PE, and Dunn WL. The histochemical demonstrations of 0-acylated sialic acid in gastrointestinal mucins. J Histochem Cytochem 22 : 826-831, 1974. Katsuyama T, Ota H, lshii K, et a/. Histochemical characterization of gastric mucin-secreting cells and the surface mucus gel layer. In Kasuya Y, Tsuchiya M, Nagao M, et a/., eds. Gastrointestinal Function. Regulation and Disturbances, Vol. 9. Excerpta Medica, Amsterdam, 1991 : 145-165. Suganuma T, Katsuyama T, Tsukahara M, et a/. Comparative histochemical study of alimentary tracts with special reference to the mucous neck cells of the stomach. Am J Anat 161 : 219-238, 1981. Akamatsu T and Katsuyama T. Histochemical demonstration of mucins in the intramucosal laminated structure of human gastric signet ring cell carcinoma and its relation to submucosal invasion. Histochem J 22 : 416-425, 1990. Ota H, Katsuyama T, lshii K, ef a/. A dual staining method for identifying mucins of different gastric epithelial mucous cells. Histochem J 2 3 : 22-28, 1991. Culling CFA, Reid PE, and Worth AW. Carbohydrate markers in the lower gastrointestinal tract. Methods Archiv Exp Pathol 10: 73-100, 1981. Lapertosa G, Baracchini P, Fulcheri E, and Tanzi R. 0 acylated sialic acid variants in mucinous tumors of the ovary. Virchows Arch [A] 408: 395-402, 1986. Bara J, Malarewicz A, Loisllier F, ef a/. Antigens common to human ovarian mucinous cyst fluid and gastric mucosa. Br J Cancer 36: 49-56, 1977. Fox H, Kazzaz B, and Langley FA. Argyrophil and argentaffin cells in the female genital tract and in ovarian mucinous cysts. J Pathol 88: 479-488, 1964. Satake T, Matsuyama M, Kazuya K, Suchi T, and Sat0 T. Argyrophil reactive cells in the normal uterus and differentiated endometrial adenocarcinoma. Acta Pathol Jpn 3 2 : 1017-1026, 1982. Satake T and Matsuyama M. Argyrophil cells in normal endometrial glands. Virchows Arch [A] 410: 449-454, 1987.
0 1992
The Japanese Society of Pathology
HistochemicaI Demonst rat io n of Ga st ro intest ina I M ucins in Ovarian Mucinous Cystadenoma
Tanri Shiozawa1*2,Yoshiharu Tsukahara', Keiko Ishi?, Hiroyoshi Ota2, Jun Nakayama2, and Tsutomu Katsuyama2
Forty-one specimens of mucinous cystadenoma (MCA) of the ovary and 13 specimens of normal uterine endocervix were investigated histochemically using alcian blue pH 2.5PAS, high iron diamine-alcian blue pH 2.5, galactose oxidase-Schiff reaction (GOS, for detection of gastric surface mucous cell mucins), paradoxical Concanavalin A staining (PCS, for detection of gastric gland cell mucins) and periodic acid-sodium borohydride-potassium hydroxide-PAS (PA-SB-PH-PAS) reaction (for detection of mucins of the large intestine). In addition, Grimelius staining was carried out to explore the distribution of endocrine cells. The MCAs contained abundant neutral mucins but a little acid mucins, whereas sulfomucins predominated i n normal endocervices. Among the MCAs, 30,26,2 and 9 had tumor cells positive for GOS, PCS, PASB-PH-PAS and Grimelius stain, respectively. These results indicate that MCAs frequently contain tumor cells of the gastrointestinal type and endocrine cell type. Endocervical epithelia, on the other hand, lack reactivity for PCS, PA-SB-PH-PAS and Grimelius stain, although i n 8 of the present specimens, the lining cells showed weak GOS reactivity. Acta Pathol Jpn 42 : 104-110, 1992. Key words : Ovarian mucinous cystadenoma, Uterine cervix,
Mucin, Grimelius stain, Histochemistry
Mucinous cystadenoma (MCA) is a common ovarian neoplasm. Characteristically, it contains abundant epithelial mucins, and the tumor cells lining the cyst produce mucins. There has been considerable debate over the histogenesis of this tumor, and early reports referred to the teratoma (1-4) and metaplasia (5-9)
Received May 23, 1991. Accepted for publication October 14, 1991. Departments of 'Gynecology and Obstetrics, and 'Laboratory Medicine, Shinshu University School of Medicine, Matsumoto. Mailing address: Tsutomu Katsuyama, M.D., Ph.D., Department of Laboratory Medicine, Shinshu University School of Medicine, Asahi 3-1-1, Matsumoto 390, Japan.
theories. In this study, we attempted to characterize the tumor cells of MCA using histochemical techniques for differentiation of mucins. In addition, uterine endocervical mucous cells were examined by the same techniques in order to compare their histochemical properties with those of the tumor cells. The results indicated that most MCAs contained mucous cells characteristic of the gastrointestinal mucosa. The mucins in MCAs were found to differ from those in endocervical cells, which lacked the properties characteristic of gastrointestinal m uci ns.
MATERIALS AND METHODS Resected tumor specimens were obtained from 41 patients with MCA of the ovary. The patients ranged in age from 11 to 81 yr, with a mean of 42.3yr. In addition, 13 uterine cervices were examined. These were obtained from uteri removed because of leiomyoma, and were histologically normal. They included 8 specimens in the proliferative, 3 in the secretory and 2 in the postmenopausal phases. All tissues were fixed in buffered 10% formalin and em bedded in paraffin. T hree-micro meter-t hic k seria I sections were prepared and stained with hematoxylin and eosin (HE). In addition, they were stained with alcian blue pH 2.5-PAS (AB-PAS) (lo), high iron diamine-alcian blue pH 2.5 (HID-AB) (1l), galactose oxidaseSchiff (GOS) (12), paradoxical Concanavalin A staining (PCS) (13) and periodic acid-sodium borohydride-potassium hydroxide-periodic acid-Schiff (PA-SB-PH-PAS) (14). Adiacent tissue sections were also stained by the Grimelius technique. The histochemical specificities of these methods and the sources of the reagents are listed in Table 1.
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Table 1. Histochemical Methods Employed and Their Specificities
Method (abbreviation) Alcian blue pH 2.5-PAS (A B- PAS) High iron djamine-alcian blue pH 2.5 (HI D-AB) Galactose oxidase-Schiff (GOS) Paradoxical Concanavalin A staining (PCS)a Periodic acid-sodium borohydride-potassium hydroxide-PAS (PA-SB-PH-PAS) Grimelius stain
Specificity Detects mucins and differentiates acid and neutral mucins Differentiates sialo- and sulfomucins Identifies mucins of gastric surface mucous cells Identifies mucins of gastric gland mucous cells Identifies sialic acid with 0-acetyl group(s) on C,-,-,, characteristic for goblet cells of large intestine Identifies endocrine cells
Reagent m -dia mineb p-diarninec Galactose oxidasec Concanavalin Ac
This table is modified from that in the report by Akamatsu and Katsuyama (17). a By PCS, epithelial mucins are classified into three groups. In this study, only the sequence for identifying class Ill rnucins, which is a hallmark of gastric gland mucous cells, was carried out. b Kanto Chemicals, Tokyo, Japan. C Sigma, St. Louis, Missouri, USA.
RESULTS Histology The 41 cases of MCA included 34 benign cases and 7 cases containing lesions of borderline malignancy (Table 2). The tumor cells were mostly columnar cells containing abundant mucins in their apical cytoplasm (Fig. la). Cuboidal or columnar cells with faintly eosinophilic cytoplasm, which contained little mucosubstances, and goblet cell-type tumor cells were seen less frequently (Fig. 1b). Endocrine cells and Paneth cells were not clearly evident by HE staining. The first type corresponded to the so-called endocervical cells described by Fenoglio et a/. (8). Their “intestinal-type’’ epithelial cells consisted of goblet cells and argyrophil cells intermingled with “absorptive cells’’, compatible with the latter cell group in our study. Fenoglio et a/. (8, 9) classified MCA into three groups. When the lining epithelium was composed of a monotonous arrangement of mucin-filled columnar cells with basal nuclei, MCA was classified as the endocervical type. On the other hand, when the lining epithelium consisted of absorptive cells, goblet cells and argyrophil cells, the MCA was classified as the intestinal type. MCA was classified as being of mixed type if the tumor contained both intestinal- and endocervical-type cells. Table 2. Classification of Ovarian Mucous Cystadenomas Examined
Benign Borderline malignancy
Endocervical tvoe Mixed tvPe 23 11 3 4
Tumors showing borderline malignancy were also classified following these criteria (Figs. l c , d). Twentysix of the cases examined in the present study were of the endocervical type, and 15 were of the mixed type. None were classified as belonging to the intestinal type. Three cases also had a dermoid cyst, but Brenner nests were not found in any of them. In about half of the cases examined, the tumor cells occasionally formed gland-like structures (Fig. le). They had clearer cytoplasm than ordinary endocervicaltype tumor cells, and in adjacent sections, these cells occasionally stained for class Ill mucins. In normal uterine cervices, the lining mucous cells resembled mucous cell-type tumor cells, but had a smaller amount of cytoplasm (Fig. lf). They did not show significant morphological changes between the two menstrual phases, or between the reproductive and menopausal states. Histochemistry Results obtained by histochemical staining are summarized in Table 3.
AB-PAS : In MCAs, endocervical-type tumor cells exhibited intense PAS reactivity, but mostly showed absence of, or only faint alcianophilia (Fig. 2a). In cases showing borderline malignancy, intracytoplasmic mucins tended to be reduced, and cell coat-type mucins, which present on the apical surface, were more evident (Fig. 2b). However, the acidity of mucins was consistent with ordinary endocervical-type tumor cells. Goblet cell-type tumor cells revealed intense alcianophilia, and “absorptive cells” possessed surface coats (Fig. 2c). In the normal endocervices, mucous cells lining either
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Mucins of Ovarian Mucinous Cystadenoma (Shiozawa et a/.)
Figure 1. Histology of mucinous cystadenoma (MCA). HE. a : Endocervical-type mucinous cystadenoma (MCA). Note orderly array of tall columnar cells with basal nuclei. b : Mixed-type MCA. Goblet cells (arrows) are intermingled with “absorptive cells”. c : Endocervical type with borderline malignancy. Slight cellular atypia is evident. d : Mixed type with borderline malignancy. Note slight nuclear atypia. Arrows indicate goblet cell-type tumor cells. e : Endocervical type. Gland-like indented portions are evident. cf. Figure 3a. f : Normal endocervical mucous cells. The cytoplasm is generally smaller in extent than that of endocervical-type tumor cells.
the cervical surface or clefts showed intense staining for acid mucins (Fig. 2d). The acidity was consistent, irrespective of sexual phase.
HID-AB : In MCAs, acid mucins were detected in 38 cases. A l l specimens had sialomucins, which predominated in 35 cases. Alcianophilia of endocentical-
type tumor cells was, however, much less marked than that of endocervical lining cells (Fig. 2e), and occasionally no staining was seen. Twenty cases had sulfomucins, which predominated in only 3 cases. All 3 of the cases which lacked acid mucins were of the benign endocervical type. Goblet cell-type tumor cells consistently showed alcianophilia, indicating the presence of
Figure 2. Histochemical reactivities of MCAs and endocervical epithelia. Figures 2a to 2 d were prepared from tissue sections stained with AB pH 2.5-PAS, Figures 2e t o 2g from tissue sections stained with HID-AB pH 2.5 and Figures 2h t o 2 j from tissue sections stained with GOS reaction. a : Endocervical-type MCA. Tumor cells reveal intense PAS reactivity. b : Endocervicaltype MCA with borderline malignancy. lntracytoplasmic mucins are almost absent. Counterstained with hematoxylin. c : Mixed-type MCA. Goblet cells show intense alcianophilia. Note the surface coat on the absorptive cells. d : Normal endocervical mucous cells. These cells show intense alcianophilia. e : Endocervical-type MCA. Only faint or negligible alcianophilia IS evident. f : Mixed-type MCA. Goblet cells reveal intense alcianophilia. g : Normal endocervical mucous cells. Marked HID reactivity is noted. h : Endocervical-type MCA. Tall columnar cells show intense reactivity. i : Mixed-type MCA. Goblet cells (arrows) lack reactivity. j : Normal endocervical mucous cells. These cells are also GOS-reactive, but stain only faintly. Counterstained with hematoxylin.
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Table 3. Histochemical Reactivities of Mucous Cells in Ovarian Mucous Cystadenoma
AB-PASa R>B R=B Rsulfo sialo = sulfo sialo 50% 50-10% B means that PAS reactivity of tumor cells predominates compared with their alcianophilia, but includes cases which lack alcianophilia. R < B means that alcianophilia of tumor cells predominates and includes those in which all tumor cells possess alcianophilia. b sialo>sulfo means that sialomucin is more abundant than sulfomucin, but includes cases which lack sulfomucin. There were no cases which contained sulfomucin only.
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Figure 3. Other histochemical reactivities of MCA. a : Endocervical-type MCA. Prepared from the section adjacent to that used for Figure 2. Basal regions (arrows) of gland-like portions stain for class Ill mucins. PCS. b : Mixed-type MCA. Goblet cells contain 8-0-acetylated sialic acid. PA-SB-PH-PAS. c : Mixed-type MCA. Endocrine cells (arrows) are intermingled with “absorptive cells”. Grimelius stain.
sialomucins (Fig. 2f). On the other hand, sulfomucins were distinctly dominant in the endocervical lining cells (Fig. 2g). GOS: In 30cases GOS-reactive tumor cells were demonstrated. Columnar cells showing intense PAS reactivity were stained distinctly with this method (Fig. 2h). In 18 of 30 cases, 10-50% of tumor cells showed GOS reactivity. An incidence of over 50% was found in only 2 cases. No correlation was noted between atypia and GOS reactivity. Goblet cells lacked GOS reactivity almost entirely (Fig. 2i). Among 13 normal endocervical specimens, 8 were positive for the GOS reaction, although the intensity of staining was much lower than that of GOS-reactive tumor cells of MCAs (Fig. 2j).
PCS : Columnar cells containing class Ill mucins were demonstrated in 2 6 cases, which included 1 7 cases of the endocervical type and 9 of the mixed type. The positive cells usually occurred in the indented regions of gland-like portions, although they were also found among the ordinary endocervical-type tumor cells (Fig. 3a). Their incidence in each case was only a few percent at maximum. Compared with adjacent sections stained with AB-PAS or HID-AB, positive cells were mostly stained for neutral or weakly acidic mucins. Strongly acidic cells never revealed class Ill mucins. Among 2 6
positive cases, 2 2 also contained GOS-reactive cells. Class Ill mucin-containing cells showed weak or negligible GOS reactivity, and vice versa. Tumor cells showing borderline malignancy lacked class Ill reactivity. In normal endocervices, no lining cells showed class Ill reactivity. PA-SB-PH-PAS : Among 41 cases, only 2 contained tumor cells showing reactivity for 8-0-acetylated sialic acid. All of these were of the goblet cell type (Fig. 3b). In normal endocervices, no lining cells stained for 8-0acetylated sialic acid. Grimelius stain : Argyrophil cells were demonstrated in 9 cases (Fig. 3c). There was no relation between the distribution of argyrophil cells and other types of tumor cell. No endocervical epithelia showed this reaction.
DISCUSSION One of the most striking features of MCA is its ability to synthesize and secrete mucins, a property that gives the cystic fluid its characteristic mucinous appearance. Characterization of these mucins would provide information to help clarify the histogenesis of this tumor. Tumor cells responsible for retention of mucins can be classified into two types; the endocervical type and goblet cell type. The former type is the predominant
Acta Pathologica Japonica 42 (2) : 1992
one, resembling mucous cells lining the uterine endocervix in possessing high columnar cytoplasm filled with mucins. The latter resembles the goblet cells present in the gastrointestinal tract, as well as those in the respiratory tract. Histochemical studies on ”endocervicaltype” tumor cells, however, indicated that the tumor cells frequently contained gastric-type mucins. Thus, with regard to mucin acidity, endocervical-type tumor cells resembled gastric surface mucous cells. In normal human tissues, covering epithelia consisting exclusively of columnar mucous cells are found only in the stomach and endocervix. The two epithelia can hardly be distinguished in ordinary preparations stained with HE. Gastric surface mucous cells, however, are characterized by a content of neutral mucins almost exclusively (15), whereas other covering epithelia of the mucous cell type consistently stain for either sialomucins or sulfomucins, as exemplified by goblet cells of the respiratory and gastrointestinal tracts, and columnar epithelia lining the pancreatic ducts and biliary tract. As confirmed in this study, endocervical mucous cells also contained highly acidic mucins, and therefore differed from “endocervical-type” tumor cells. The present study also demonstrated that some of the endocervical-type cells contained class Ill mucins. These cells were usually located in the indented portions, or lined gland-like structures in the stroma. In all vertebrates examined previously, class Ill mucins, which are specifically demonstrated by PCS, have been found only in the cardiac glands, mucous neck cells, pyloric glands and Brunner‘s glands (10, 15, 16). Class Ill Con A reactivity is a useful marker for identifying these cells, and its presence indicates that the cells containing these mucins have properties similar t o those of the glandular epithelia described above. GOS reactivity of the endocervical-type tumor cells resembled that of gastric surface mucous cells. In the gastrointestinal and respiratory tracts, gastric surface mucous cells show the most intense GOS reactivity (10, 15, 17). Endocervical-type tumor cells also showed relatively intense GOS reactivity, whereas the endocervical mucous cells stained rather faintly. It is noteworthy that in both MCAs and the stomach, mucous cells showing intense GOS reactivity lack class Ill Con A reactivity (18). However, GOS-reactive tumor cells were demonstrated in only 30 of 41 cases. In the human stomach, the surface mucous cells, which exhibit intense blood group activity, may show a rather weak GOS reaction(l8). This phenomenon could be due to the fact that galactose oxidase selectively modifies the p-anomer of galactose and N-acetylgalactosamine, whereas the a-anomer of these two sugar residues occupies the non-reducing terminal of blood group B and
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A determinants, respectively. Although blood group activity of tumor cells in MCAs was not examined in this study, endocervical-type tumor cells which showed weak GOS reactivity might contain mucins with blood group activity. The present observations indicated that so-called endocervical-type tumor cells included gastric-type mucous cells, but did not exclude the possible existence of real endocervical-type tumor cells in MCAs. A specific marker for the latter cell type is a prerequisite for demonstration of its presence in MCAs. 8-0-Acetylated sialic acid was demonstrated in two cases. Among various human organs, 8-0-acetylated sialic acid is most abundant in goblet cells of the large intestine (14), and the PA-SB-PH-PAS sequence has been employed to detect metastatic bowel carcinoma (19). Lapertosa et a/. (20) found this sialic acid in goblet cell-type tumor cells of MCAs in over 50% of cases examined. The difference in the incidence of positive goblet cells between our study and that of Lapertosa et a/. might reflect the number of blocks examined in each case. Since most goblet cell-type tumor cells contained ordinary sialic acid, these cells might possess properties of small-intestinal goblet cells. By immunizing rabbits with high-molecular-weight proteins (HMWP) purified from gastric mucosa and the contents of MCAs, Bara et a/. (21) obtained antisera against gastric HMWP and MCA HMWP, respectively. Both of the antisera reacted with both gastric mucosa and MCA, indicating the presence of macromolecules bearing epitopes common to these two tissues. Their results also suggested the presence of antigens common to the gastric mucosa, colon mucosa and MCA, and agree well with our findings. Bara et a/., however, did not refer to the location of the epitopes responsible for these reactions, i.e. on the peptide portions or saccharide portions of the macromolecules purified. As confirmed in the present study, endocrine cells have been noted in MCA. Recent studies have shown that APUD cells occur more widely in human tissues than believed previously. Although we could not identify argyrophil cells in the endocervix, Fox et a/. (22) and Satake et a/. (23) noted their presence a t the same site. Moreover, Satake and Matsuyama (24) found endocrine cells in the endometrium and noted that they contained serotonin and somatostatin. The demonstration of endocrine cells, therefore, would not be a hallmark of endoderma I a nd gas t rointest ina I epithelia I origin. Several theories have been proposed for the histogenesis of MCA, including the teratoma (1-4) and metaplasia (5-8) theories. The latter asserts that all types of tumor cell are derived from metaplasia of the coelomic epithelium, whereas, in the former, the mucous
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cells of MCA are explained as being due t o clonal proliferation of a particular cell species, a s exemplified by struma ovarii. However, neither intestinal metaplasia nor gastric metaplasia has been observed in the mesothelium or the female genital tract. Moreover, tumor cells showing a gastric phenotype have never been found in carcinomas of the female genital tract except for cystadenocarcinoma of the ovary (Shiozawa and Katsuyama, unpublished observation). In teratoma, o n the other hand, gastrointestinal epithelia are frequently a major constituent. Thus the teratoma theory seems better fitted t o our present results.
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