Path. Res. I'mct. 187,472-476 (1991 )
Small-Cell Carcinoma of Gallbladder An Immunocytochemical and Ultrastructural Study A. O. Cavazzana, A. S. Fassina, M. Tollot and V. Ninfo Istituto di Anatomia ed Istologia Patologica della Universita di Padova, Padova, Italy
SUMMARY An unusual carcinoma of the gallbladder in a seventy·one·year·old woman displayed features of a well·differentiated adenocarcinoma, atypical carcinoid and small cell undifferentiated carcinoma. The patient died from progressive hepatic failure four months after surgery. Autopsy showed bulky liver masses and several peritoneal nodules exclusively composed of small, hyperchromatic cells. The neuroendocrine nature of the small cell component of the tumor was documented by the presence of neurosecretory granules at the ultrastructuralleve! and by immunocytochemical positivity to NSE and SynalJtophysin. The epithelial markers, cytokeratin and CEA, were also positive in the carcinoid and in the undifferentiated portions of the tumor. A common endodermal origin is suggested for carcinoid and small cell carcinoma of the gallbladder.
Introduction Small cell carcinoma of the gallbladder is a rare lesion, representing about 4% of all carcinomas in this organ 29 . Predominantly a disease of older women with a clinical history of stones, these tumors show an aggressive clinical course and dearh usually occurs within a few months of the diagnosis'. Only four cases of small cell carcinoma associated with a well·differentiated adenocarcinoma have been previously described 29 • This report illustrates the immunocytochemical and ultrastructural features of small cell carcinoma, and addresses its histogenetic relationship with neuroendocrine tumors and the more common adenocarcinoma of the gallbladder. Case Report A 71·ycar·old woman was hospitalized for extrasys· toles; during her stay she developed a severe obstructive jaundice. Laboratory data showed an elevated total biliru· bin (262.6 UmollL) and a slight increase in carcinoem· bryonic antigen (CEA) (13 U). Hepatic echography and abdominal tomography revealed the presence of a 30 X 52 mm mass involving the common bile duct. 0344-0338/91/0187-0472$3.50/0
Cholecystectomy was performed, and the hepatic hilar lymph nodes were excised. Four months after surgery, the patient was again hospitalized for severe jaundice, and died a few days after admission with progressive hepatic failure.
Post-mortem examination disclosed a conspicuous
hemorrhagic ascites with many peritoneal nodules. The liver was extensively involved (liver weight 1300 g) by voluminous, centrally necrotic and superficially ulcerated masses, measuring from 5 to 10 em in diameter. Mesenteric, aortic and mediastinal lymph nodes were free of metastases. There was no evidence of metastatic disease or other primaries in lungs, small and large intestine, pan· creas, ovaries and uterus. Material and Methods Formalin-fixed, paraffin-embedded tissues were available for histochemical, immunocytochemical and ultrastructural analysis. Five micron thick paraffin sections were stained according to the H & E, Periodic acid-Schiff (PAS ), diastase-PAS, Aleian Blue and Grimelius methods. Immunocytochemical analysis was per-
formed using the Avidin-Biotin- Complex (ABC), technique as
previously described lt , employing a Vectastain ABC kit (Vector Lab, Burlingame, California, USA). The presence of the following © 1991 by Gustav
Fisch~r V~r1ag,
Stuttgart
Small-Cell Carcinoma of Gallbladder· 473
antigens was investigated: cytokeratin PKK 1 (1 : 10, Labsystem, Finland); CEA (1: 400, Dakopatts, Copenhagen, Denmark );
chromogranin A (1 : 100, Boehringer Mannheim, \Vest Germany); synaptophysin (5 micrograms/ml, Boehringer Mannheim, West Germany), and neuron specific enolase (NSE, 1: 200, Dakopatts), Normal large bowel sections were used as positive controls; in the same sections primary antibodies were omitted for negative control. Formalin-fixed, paraffin-embedded material was also deparaffinized and processed for electron microscopy by routine techniques l2 .
Results The gallbladder was enlarged and showed a whitegreyish mass of 5 X 3 X 3 em involving the cystic and common hepatic duct. The lumen was filled by dense bile and no stones were observed.
Fig. 2. Glandular lum ens were lined by epithelial cells gradually merging into nodules composed of small round cells (a) ( H& E, X 250). Cells with pale cyroplasm, hyperchromatic round or oval nuclei arranged in a lobular fashion were also observed in the neoplasia (b) (H & E, x 400).
Fig. 1. A well-differentiated adenocarcinoma of the gallbladder (top left) is associated with a diffuse proliferation of small round
cells (bottom right) (H & E,
X
200 ).
Microscopic examination of the mass revealed a welldifferentiated adenocarcinoma admixed with a diffuse proliferation of small round cells and nests of atypical carcinoid (Fig. 1). The undifferentiated component consisted of round andlor spindle cells with hyperchromatic nuclei and a rim of scanty and poorly defined cytoplasm. The cells were arranged in solid sheets that infiltrated the hepatic parenchyma. Transitional features of well-differentiated carcinoma and small cell carcinoma were occasionally noticed (Fig. 2a). Solid nests of round cells with palc eosinophilic cytoplasm and slightly irregular, hyperchromatic nuclei and small nucleoli, reminiscent of atypical carcinoid (Fig. 2 b), were also present. The luminal portion of the neoplasia was composed of well-differentiated adenocarcinoma. The hepatic hilar lymph nodes showed microscopic foci of metastatic adenocarcinoma.
474 . A. O. Cavazzana er al. Histochemical srudies demonstrated PAS positive, diastase-resistent and Aleian Blue positive material exclusively in the adenocarcinomatous portion of the tumor. Grimelius-positive granules were derecred only in the cytoplasm of the undifferentiared small cells and in rhe atypical carcinoid areas (Fig. 4, insert). No features of intestinal metaplasia were encountered in the normal mucosa adjacent ro the tumor. Immunocytochemical invesrigation disclosed thar cyrokeratin and CEA were strongly positive in the welldifferentiated adenocarcinoma and focally in the other two neoplastic components (Fig. 3a ). CEA immunoreactivity varied in the different portions of rhe tumor. The apical border of cells lining the neoplastic glands was heavily decorated by anti-CEA antibody, while a diffuse cytoplasmic CEA reactivity was detecred in the small cell component. NSE and synaptophysin were positive only in the carcinoid and undifferentiated small cells (Fig. 3 b), while chromogranin was negative in all portions of the tumor.
Fig. 4. Small cell carcinoma seen ar E.M. (x 6000). The Grime·
lius positive granuJcs detected at the light microscope (upper left
insert, x 400) were still well recognizable ultrastructurally (lower left insert, x 15000).
Ultrastructurally, the small cell carcinoma was composed of a uniform population of round or oval cells, characterized by a few electron-dense granules, bundles of intermediate filaments, and rare primitive intercellular
a
junctions (Fig. 4). Microscopic evaluation of the liver and peritoneal nodules found at autopsy, revealed a uniform population of small cells with dark hyperchromatic nuclei, arranged in ribbons, strands and small solid nests, with no evidence of glandular differentiation. The nodules displayed diffuse cytoplasmic positivity to cytokeratin, NSE and synaptophysin. Discussion
Fig. 3. Evident perinuclear positivity
to allti-cytokerarin anti· bodies was presenr in the small cell carcinoma (a) (ABC-method,
x 400) as well as a st rong cytoplasmic positivity to anti-
Synaptophysin (b) (ABC-method,
X
400).
Small cell carcinomas of the gasrro-intcstinal traer are rare tumors generally occurring in elderly individuals, with an unfavorable prognosis, regardless of their site of origin and size 1,9, 16,2S Histologically, these tumors are made up of sheets, cords or festoons of uniform small round cells with hyperchromatic nuclei, inconspicuous nucleoli and scant cytoplasm. Fusiform cells with more abundant cytoplasm are also observed',.lo. The origin of small-cell
Small-Cell Carcinoma of Gallbladdcr . 475 carcinomas, regardless of their location, is still controversial, and an undifferentiated endodermal 4 or neuroendocrine origin has been alternatively proposed2l • The presence of neurosecretory granules at the ultrastructurallcvcl 9 , 16 and positivity to neuroendocrine markers, such as NSE, LEU 7, neurofilaments, bombesin and chromogranin 6,13,3" strongly support a neuroendocrine origin for this tumor. According to Paladugu, neuroendocrine tumors of the lung present a spectrum of overlapping morphologic features, ranging from typical carcinoid to small cell carcinoma. Increased mitotic activity, single cell necrosis, nuclear pleomorphism and hyperchromasia identify the atypical carcinoid as an intermediate form in the spectrum. The collective name of "Kulchitzky Cell Carcinoma" was then proposed for these tumors"). The origin of neuroendocrine cells has been extensively revised in recent years, and a common neuroectodermal origin for all cells belonging to the A.P.U.D. system 22 is now accepted with skepticism. Experimental animal models seem to support an endodermal origin for at least some of these cells s,24, and therefore the endodermal stem cell has been advanced as the common precursor of both epithelial and endocrine intestinal cells. Moreover, the co-expression of markers typical of embryonic intestinal (CEA), epithelial (cytokeratin) and neuroendocrine cells (NSE, bombesin and chromogranin) in human neuroendocrine tumors has been offered as supportive evidence of the endodermal origin of these tumors 17. This thesis seems further sustained by the occurrence of so-called "composite carcinoma-carcinoid" tumors of the gastro-intestinal tract 14,.12. These rare tumors, in fact, show both typical adenocarcinoma and carcinoid features. Carcinoids microscopically resembling a well-differentiated adenocarcinoma have already been reported in different organs, including the gallbladder I', 19,26. Furthermore, squamous, glandular and neuroendocrine differentiation were shown in small cell carcinomas of the lung and gastro-intestinal tract IS , 23. All these findings point to an intriguing histogenetic link between epithelial cells, neurosecretory cells and small-cell carcinoma. The gallbladder constitutes a unique model to study this problem, since neuroendocrine cells arc absent in the normal cholecystic mucosa2, 29. This partially explains the rarity of neuroendocrine tumors in this organ; in a series of 2837 carcinoid tumors only one was located in the gallbladder7 • Nevertheless, neuroendocrine cells may be found in the context of a metaplastic cholecystic mucosa. An intestinal type of metaplasia is known to occur in chronic conditions, such as cholelithiasis and chronic cholecystitis, and an entero-endocrine differentiation was observed in well-differentiated adenocarcinoma'. Metaplastic neuroendocrine changes in the non-neoplastic mucosa, in fact, were previously described in endocrine cell tumors admixed with adenocarcinoma of the gallbladder' 7,28. In our case, the absence of both metaplastic changes in the remaining normal mucosa and endocrine differentiation in the adenocarcinomatous portion of the tumor suggested a divergent, epithelial and neuroendocrine differentiation of the neoplastic cells. This hypothesis was further endorsed by the presence of transition areas
between the different components of the tumor. Areas of well-differentiated adenocarcinoma were intermingled with nests of pale, slightly eosinophilic cells with hyperchromatic and pleomorphic nuclei, and diffuse sheets of small, dark, occasionally fusiform cells, representative of atypical carcinoid and small cell carcinoma, respectively. The positivity to CEA and cytokeratin in gastrointestinal tumors is considered supportive evidence for the endodermal origin of the neoplasia 3, 8, whereas NSE and synaptophysin are the two most reliable markers for neuroendocrine differentiation in gastrointestinal tumors. The majority of gastro-intestinal carcinoids have, in fact, been reported positive for these two markcrs 10 We observed a strong cytoplasmic positivity for NSE and synaptophysin in the small cell and carcinoid areas of the tumor. This finding confirmed the neuroendocrine nature of the small undifferentiated cells, which was further verified by argyrophilia and the ultrastructural observation of neurosecretory granules. Moreover, scattered cells in both the carcinoid and small cell portions of the tumor showed positive reactions with anti-CEA and anti-keratin antibodies. Our immunocytochemical findings and the above described transitional microscopic features appear to provide further evidence for a divergent epithelial-neuroendocrine differentiation of the endodermal stem cell. Acknowledgement We ar~ in debt with Prof. M. Rugge for critical reviewing the manllscnpt.
References 1 Albores-Saavedra j, Soriano ], Larraza-Hernandez 0, Aguirre J, Heuson DE (1984) Oat cell carcinoma of the gallbladder. Hum Pathol15: 639-646 2 Albores-Saavedra J, Henson DE, Angeles-Angeles A (1989) Entero endocrine cell differentiation in carcinoma of the gallbladder and mucinous cystoadenocarcinomas of the pancreas. Path Res Pract 183: 169-175 :1
Altmannsberger M, Weber K, Holscher A, Schauer A,
Osborne M (1982) Antibodies
to
intermediate filaments as
diagnostic tools: human gastrointestinal carcinomas express prekeratin. Lab Invest 46: S20-S26
4 Banks-Schlegel SP, Gadzar AF, Harris CC (1985) intcrmediate filaments and cross-linked envelope expression in human lung tumor cell lines. Cancer Res 45: J J87-1197 5 Cox WF Jr, Pierce GB (1982) The endodennal origin of the endocrine cell of an adenocarcinoma of the colon of the rat. Cancer 50: 1530-1538 6 Gadzar AF, Carncy DN, Nau MM, Minna JD (1985 ) Characterization of variant subclasses of cell lines derived from small cell lung carcinoma having distinctive biochemical, morphological and growth properties. Callcer Res 45: 2924-2930 7 Godwin JD II (1975) Carcinoid tumors: an analysis of 2837 cases. Cancer 36: 560-569 II Gold P, freedman SO (1965 ) Specific carcinoembryonic antigens of the human digestive system. ] Exp Mcd J22: 467-481
476 . A. O. Cavazzana et al.
, Gould VE, Chejfec G (1978) Neuroendocrine carcinomas of the colon. Am J Surg Pathol 7: 31-38 10 Gould VE, Lee 11Wiedenmann B, Moll R, Chejfcc G, Franke WW (1986) Synaproph isin: A novel marker for neurons, certain neuroendocrine cells and their neoplasms. Hum Parhol 17: 979-983 11 Hsu SM, Raine L, Fanger H (1981 ) Use of the AvidinBiotin ~ Peroxidasc complex (ABC) in immunoperox ida se technique: A comparison between ABC and un labelled antibody (PAP) procedures. J Histochem Cytochem 29: 577-580 12 Hultquist T, Karlsson U (1972) Use of formalin-fixed, paraffin-embedded biopsy or autopsy material for electron microscopy. Path Europ Vol 2: 97-101 13 Leth VP, Stenman S, Miettinen M, Dahl D, Virtancn I (1983) Expression of a neural type of intermediate filament as a distinguishing feature between oat cell carcinoma and the lung cancers. Am J Pathol 110: 113- 118 14 KJappenbach RS, Kurman RJ, Sinclair CF,Jarncs LP (1985) Composite carcinoma-carcinoid rumors of [he gastrointestinal tract. A morphologic, histochemical and immunocytochemical study. Am J Cli n Pathol 84: 137-143 15 McDowell EM, Trump BF (1981 ) Pulmonary small cell carcinoma s howing tripartite differentiation in individual cells. Hum Pathol 12: 286-294 16 Mills SE, Allen MS, Cohen AR (1983) Small cell undiffcrentiated carcinoma of the colon. A clinicopathological srudy of five cases and their association with colonic adenomas. Am JSurg Pathol 7: 643-651 17 Nash SV, Said JW (1986) Gastroenteropancreati c neuroendocrine tumors. A histochemical and immunohistochemical study of epithelial (keratin proteins, carcinoembryonic antigen) and neuroendocrine (neuron specific enolase, bombesin and chromogranin) markers in foregut, midgut and hindgut rumors. Am JClin Pathol 86: 415-422 18 Noda M, Miwa A, Kitagawa M (1989 ) Carcinoid rumors of the ga llbladder with adenocarcinomatous differentiation : amorphologic and immunohistochemica l srudy. Am J Gasrrocnterol 84: 953-957 19 Ohtsuki M, Midorikawa 0, Okada H, Morikawa 5, Sakaguchi M (1989) Pulmonary atypical carci noid tumor with markcd alpha fetoprotein production and features of an adenocarcinoma differentiation. Path Res Pract 184: 86-94
20 Paladugu RR, Benfield Jr, Pak HJ, Ross RK, Teplitz RL (1985) Bronchopulmonary Kulchitzky cell carci nomas: a new classification for typical and atypical carcinoids. Cancer 55: 1303-1311 21 Pearse AG (1969) The cytochemistry and ultrastructure 01 polypeptide hormone producing cells of the APUD series and the embryonic, physiologic and pathologic implications of the concept. J Hisrochem Cytochem 17: 303- 313 22 Pearse AG, Polak JM (1971) Neural crest origin of the endocrine polypeptide (A PUD) cells of the gastrointestinal tract and pancreas. Gut 12: 783-788 2J Petrelli M, Tetangco E, Reid JD (198 1) Carcinoma of the colon with undifferenriated, carcinoid, and squamous cell features. Am J Clin Pathol 75: 581-584 24 Pictet RL, Rail LB, Phelps P, Runer WJ (1976) The neutral crest and the origin of insulin-producing and other gastrointestinal hormone· producing cells. Science 191: 191-1 92 25 Schwartz AM, Orenstein JM (1985 ) Small cell undiffercnriated carcinoma of the rectosigmoid colon. Arch Pathol Lab Med 109: 629-632 26 Soga J, Tazawa K, Aizawa 0, Wada K, Tuto T (1971 ): Argentaffin cell adenocarcin oma of the stomach: an atypical carcinoid ' Cancer 33: 999-1003 27 Yamamoto M, Nakajo S, Miyoshi N, Nakai 5, Tahara E (1989): Endocrine Cell Carcinoma (Carcin oid) of the Gallbladder. Am J Surg Pathol 13 (4): 292-302 211 Wada A, Ishiguro 5, Tateishi R, Ishikawa 0, Matsui Y (1983): Carcinoid tumor of the gallbladder associated with adenocarcinoma. Cancer 51: 1911-191 7 29 Weedon D (1984) Pathology of the gallbladder. Masson Monographs in diagnostic pathology 3D WHO (1981) Histological Typing of Lung Tumours. II Ed. WHO Geneva 31 Wick MR, Weatherby RP, Weiland LH (1987): Small cell neuroendocrine carcinoma of the colon and rectum: Clinical, histological and ultrastrucrural study and immunohistochemical comparison with cloacogenic carcinoma. Hum Pathol 1 8: 9-2 1 J2 Wisniewski M, Toker C (1982): Composite tumor of the ga llbladder exh ibiting both carcinomatous and carcinoidal patterns. Am J. Gasrroenterol. 58: 633-637
Received January 18, 1990· Accepted in revised form August 28, 1990
Key words: Gallbladder - Adenocarcinoma - Neuroendocrine tumors Andrea 0. Cavazzana M.D., Istituto di Anatomia Patologica, Via Gabelli 6 1, 35 121 Padova, Italy
Small-Cell Carcinoma of Gallbladder An Immunocytochemical and Ultrastructural Study A. O. Cavazzana, A. S. Fassina, M. Tollot and V. Ninfo Istituto di Anatomia ed Istologia Patologica della Universita di Padova, Padova, Italy
SUMMARY An unusual carcinoma of the gallbladder in a seventy·one·year·old woman displayed features of a well·differentiated adenocarcinoma, atypical carcinoid and small cell undifferentiated carcinoma. The patient died from progressive hepatic failure four months after surgery. Autopsy showed bulky liver masses and several peritoneal nodules exclusively composed of small, hyperchromatic cells. The neuroendocrine nature of the small cell component of the tumor was documented by the presence of neurosecretory granules at the ultrastructuralleve! and by immunocytochemical positivity to NSE and SynalJtophysin. The epithelial markers, cytokeratin and CEA, were also positive in the carcinoid and in the undifferentiated portions of the tumor. A common endodermal origin is suggested for carcinoid and small cell carcinoma of the gallbladder.
Introduction Small cell carcinoma of the gallbladder is a rare lesion, representing about 4% of all carcinomas in this organ 29 . Predominantly a disease of older women with a clinical history of stones, these tumors show an aggressive clinical course and dearh usually occurs within a few months of the diagnosis'. Only four cases of small cell carcinoma associated with a well·differentiated adenocarcinoma have been previously described 29 • This report illustrates the immunocytochemical and ultrastructural features of small cell carcinoma, and addresses its histogenetic relationship with neuroendocrine tumors and the more common adenocarcinoma of the gallbladder. Case Report A 71·ycar·old woman was hospitalized for extrasys· toles; during her stay she developed a severe obstructive jaundice. Laboratory data showed an elevated total biliru· bin (262.6 UmollL) and a slight increase in carcinoem· bryonic antigen (CEA) (13 U). Hepatic echography and abdominal tomography revealed the presence of a 30 X 52 mm mass involving the common bile duct. 0344-0338/91/0187-0472$3.50/0
Cholecystectomy was performed, and the hepatic hilar lymph nodes were excised. Four months after surgery, the patient was again hospitalized for severe jaundice, and died a few days after admission with progressive hepatic failure.
Post-mortem examination disclosed a conspicuous
hemorrhagic ascites with many peritoneal nodules. The liver was extensively involved (liver weight 1300 g) by voluminous, centrally necrotic and superficially ulcerated masses, measuring from 5 to 10 em in diameter. Mesenteric, aortic and mediastinal lymph nodes were free of metastases. There was no evidence of metastatic disease or other primaries in lungs, small and large intestine, pan· creas, ovaries and uterus. Material and Methods Formalin-fixed, paraffin-embedded tissues were available for histochemical, immunocytochemical and ultrastructural analysis. Five micron thick paraffin sections were stained according to the H & E, Periodic acid-Schiff (PAS ), diastase-PAS, Aleian Blue and Grimelius methods. Immunocytochemical analysis was per-
formed using the Avidin-Biotin- Complex (ABC), technique as
previously described lt , employing a Vectastain ABC kit (Vector Lab, Burlingame, California, USA). The presence of the following © 1991 by Gustav
Fisch~r V~r1ag,
Stuttgart
Small-Cell Carcinoma of Gallbladder· 473
antigens was investigated: cytokeratin PKK 1 (1 : 10, Labsystem, Finland); CEA (1: 400, Dakopatts, Copenhagen, Denmark );
chromogranin A (1 : 100, Boehringer Mannheim, \Vest Germany); synaptophysin (5 micrograms/ml, Boehringer Mannheim, West Germany), and neuron specific enolase (NSE, 1: 200, Dakopatts), Normal large bowel sections were used as positive controls; in the same sections primary antibodies were omitted for negative control. Formalin-fixed, paraffin-embedded material was also deparaffinized and processed for electron microscopy by routine techniques l2 .
Results The gallbladder was enlarged and showed a whitegreyish mass of 5 X 3 X 3 em involving the cystic and common hepatic duct. The lumen was filled by dense bile and no stones were observed.
Fig. 2. Glandular lum ens were lined by epithelial cells gradually merging into nodules composed of small round cells (a) ( H& E, X 250). Cells with pale cyroplasm, hyperchromatic round or oval nuclei arranged in a lobular fashion were also observed in the neoplasia (b) (H & E, x 400).
Fig. 1. A well-differentiated adenocarcinoma of the gallbladder (top left) is associated with a diffuse proliferation of small round
cells (bottom right) (H & E,
X
200 ).
Microscopic examination of the mass revealed a welldifferentiated adenocarcinoma admixed with a diffuse proliferation of small round cells and nests of atypical carcinoid (Fig. 1). The undifferentiated component consisted of round andlor spindle cells with hyperchromatic nuclei and a rim of scanty and poorly defined cytoplasm. The cells were arranged in solid sheets that infiltrated the hepatic parenchyma. Transitional features of well-differentiated carcinoma and small cell carcinoma were occasionally noticed (Fig. 2a). Solid nests of round cells with palc eosinophilic cytoplasm and slightly irregular, hyperchromatic nuclei and small nucleoli, reminiscent of atypical carcinoid (Fig. 2 b), were also present. The luminal portion of the neoplasia was composed of well-differentiated adenocarcinoma. The hepatic hilar lymph nodes showed microscopic foci of metastatic adenocarcinoma.
474 . A. O. Cavazzana er al. Histochemical srudies demonstrated PAS positive, diastase-resistent and Aleian Blue positive material exclusively in the adenocarcinomatous portion of the tumor. Grimelius-positive granules were derecred only in the cytoplasm of the undifferentiared small cells and in rhe atypical carcinoid areas (Fig. 4, insert). No features of intestinal metaplasia were encountered in the normal mucosa adjacent ro the tumor. Immunocytochemical invesrigation disclosed thar cyrokeratin and CEA were strongly positive in the welldifferentiated adenocarcinoma and focally in the other two neoplastic components (Fig. 3a ). CEA immunoreactivity varied in the different portions of rhe tumor. The apical border of cells lining the neoplastic glands was heavily decorated by anti-CEA antibody, while a diffuse cytoplasmic CEA reactivity was detecred in the small cell component. NSE and synaptophysin were positive only in the carcinoid and undifferentiated small cells (Fig. 3 b), while chromogranin was negative in all portions of the tumor.
Fig. 4. Small cell carcinoma seen ar E.M. (x 6000). The Grime·
lius positive granuJcs detected at the light microscope (upper left
insert, x 400) were still well recognizable ultrastructurally (lower left insert, x 15000).
Ultrastructurally, the small cell carcinoma was composed of a uniform population of round or oval cells, characterized by a few electron-dense granules, bundles of intermediate filaments, and rare primitive intercellular
a
junctions (Fig. 4). Microscopic evaluation of the liver and peritoneal nodules found at autopsy, revealed a uniform population of small cells with dark hyperchromatic nuclei, arranged in ribbons, strands and small solid nests, with no evidence of glandular differentiation. The nodules displayed diffuse cytoplasmic positivity to cytokeratin, NSE and synaptophysin. Discussion
Fig. 3. Evident perinuclear positivity
to allti-cytokerarin anti· bodies was presenr in the small cell carcinoma (a) (ABC-method,
x 400) as well as a st rong cytoplasmic positivity to anti-
Synaptophysin (b) (ABC-method,
X
400).
Small cell carcinomas of the gasrro-intcstinal traer are rare tumors generally occurring in elderly individuals, with an unfavorable prognosis, regardless of their site of origin and size 1,9, 16,2S Histologically, these tumors are made up of sheets, cords or festoons of uniform small round cells with hyperchromatic nuclei, inconspicuous nucleoli and scant cytoplasm. Fusiform cells with more abundant cytoplasm are also observed',.lo. The origin of small-cell
Small-Cell Carcinoma of Gallbladdcr . 475 carcinomas, regardless of their location, is still controversial, and an undifferentiated endodermal 4 or neuroendocrine origin has been alternatively proposed2l • The presence of neurosecretory granules at the ultrastructurallcvcl 9 , 16 and positivity to neuroendocrine markers, such as NSE, LEU 7, neurofilaments, bombesin and chromogranin 6,13,3" strongly support a neuroendocrine origin for this tumor. According to Paladugu, neuroendocrine tumors of the lung present a spectrum of overlapping morphologic features, ranging from typical carcinoid to small cell carcinoma. Increased mitotic activity, single cell necrosis, nuclear pleomorphism and hyperchromasia identify the atypical carcinoid as an intermediate form in the spectrum. The collective name of "Kulchitzky Cell Carcinoma" was then proposed for these tumors"). The origin of neuroendocrine cells has been extensively revised in recent years, and a common neuroectodermal origin for all cells belonging to the A.P.U.D. system 22 is now accepted with skepticism. Experimental animal models seem to support an endodermal origin for at least some of these cells s,24, and therefore the endodermal stem cell has been advanced as the common precursor of both epithelial and endocrine intestinal cells. Moreover, the co-expression of markers typical of embryonic intestinal (CEA), epithelial (cytokeratin) and neuroendocrine cells (NSE, bombesin and chromogranin) in human neuroendocrine tumors has been offered as supportive evidence of the endodermal origin of these tumors 17. This thesis seems further sustained by the occurrence of so-called "composite carcinoma-carcinoid" tumors of the gastro-intestinal tract 14,.12. These rare tumors, in fact, show both typical adenocarcinoma and carcinoid features. Carcinoids microscopically resembling a well-differentiated adenocarcinoma have already been reported in different organs, including the gallbladder I', 19,26. Furthermore, squamous, glandular and neuroendocrine differentiation were shown in small cell carcinomas of the lung and gastro-intestinal tract IS , 23. All these findings point to an intriguing histogenetic link between epithelial cells, neurosecretory cells and small-cell carcinoma. The gallbladder constitutes a unique model to study this problem, since neuroendocrine cells arc absent in the normal cholecystic mucosa2, 29. This partially explains the rarity of neuroendocrine tumors in this organ; in a series of 2837 carcinoid tumors only one was located in the gallbladder7 • Nevertheless, neuroendocrine cells may be found in the context of a metaplastic cholecystic mucosa. An intestinal type of metaplasia is known to occur in chronic conditions, such as cholelithiasis and chronic cholecystitis, and an entero-endocrine differentiation was observed in well-differentiated adenocarcinoma'. Metaplastic neuroendocrine changes in the non-neoplastic mucosa, in fact, were previously described in endocrine cell tumors admixed with adenocarcinoma of the gallbladder' 7,28. In our case, the absence of both metaplastic changes in the remaining normal mucosa and endocrine differentiation in the adenocarcinomatous portion of the tumor suggested a divergent, epithelial and neuroendocrine differentiation of the neoplastic cells. This hypothesis was further endorsed by the presence of transition areas
between the different components of the tumor. Areas of well-differentiated adenocarcinoma were intermingled with nests of pale, slightly eosinophilic cells with hyperchromatic and pleomorphic nuclei, and diffuse sheets of small, dark, occasionally fusiform cells, representative of atypical carcinoid and small cell carcinoma, respectively. The positivity to CEA and cytokeratin in gastrointestinal tumors is considered supportive evidence for the endodermal origin of the neoplasia 3, 8, whereas NSE and synaptophysin are the two most reliable markers for neuroendocrine differentiation in gastrointestinal tumors. The majority of gastro-intestinal carcinoids have, in fact, been reported positive for these two markcrs 10 We observed a strong cytoplasmic positivity for NSE and synaptophysin in the small cell and carcinoid areas of the tumor. This finding confirmed the neuroendocrine nature of the small undifferentiated cells, which was further verified by argyrophilia and the ultrastructural observation of neurosecretory granules. Moreover, scattered cells in both the carcinoid and small cell portions of the tumor showed positive reactions with anti-CEA and anti-keratin antibodies. Our immunocytochemical findings and the above described transitional microscopic features appear to provide further evidence for a divergent epithelial-neuroendocrine differentiation of the endodermal stem cell. Acknowledgement We ar~ in debt with Prof. M. Rugge for critical reviewing the manllscnpt.
References 1 Albores-Saavedra j, Soriano ], Larraza-Hernandez 0, Aguirre J, Heuson DE (1984) Oat cell carcinoma of the gallbladder. Hum Pathol15: 639-646 2 Albores-Saavedra J, Henson DE, Angeles-Angeles A (1989) Entero endocrine cell differentiation in carcinoma of the gallbladder and mucinous cystoadenocarcinomas of the pancreas. Path Res Pract 183: 169-175 :1
Altmannsberger M, Weber K, Holscher A, Schauer A,
Osborne M (1982) Antibodies
to
intermediate filaments as
diagnostic tools: human gastrointestinal carcinomas express prekeratin. Lab Invest 46: S20-S26
4 Banks-Schlegel SP, Gadzar AF, Harris CC (1985) intcrmediate filaments and cross-linked envelope expression in human lung tumor cell lines. Cancer Res 45: J J87-1197 5 Cox WF Jr, Pierce GB (1982) The endodennal origin of the endocrine cell of an adenocarcinoma of the colon of the rat. Cancer 50: 1530-1538 6 Gadzar AF, Carncy DN, Nau MM, Minna JD (1985 ) Characterization of variant subclasses of cell lines derived from small cell lung carcinoma having distinctive biochemical, morphological and growth properties. Callcer Res 45: 2924-2930 7 Godwin JD II (1975) Carcinoid tumors: an analysis of 2837 cases. Cancer 36: 560-569 II Gold P, freedman SO (1965 ) Specific carcinoembryonic antigens of the human digestive system. ] Exp Mcd J22: 467-481
476 . A. O. Cavazzana et al.
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Received January 18, 1990· Accepted in revised form August 28, 1990
Key words: Gallbladder - Adenocarcinoma - Neuroendocrine tumors Andrea 0. Cavazzana M.D., Istituto di Anatomia Patologica, Via Gabelli 6 1, 35 121 Padova, Italy
