Path. Res. Pract. 187,44-49 (1991)
Human Chorionic Gonadotropin in Esophageal Carcinomas An Immunohistochemical Study I. Trias, E. Campo 1,2, C. Benasco, A. Palacin 1 and A. Cardesa Departments of Pathology, Hospital de Bellvitge "Princeps d'Espanya'; 1Hospital Clinico y Provincial de Barcelona, and 2Facultad de Medicina, Estudi General de Lleida, Universidad de Barcelona, Spain
SUMMARY We have examined immunohistochemically the presence of human chorionic gonadotrophin (hCG) in 29 esophageal carcinomas: 24 squamous cell carcinomas, 2 adenocarcinomas, 2 adenoid cystic carcinomas and 1 adenosquamous carcinoma. In hCG-positive tumors, the presence of human placental lactogen (hPL) and pregnancy-specific beta-l glycoprotein (SP-l) was also assessed. HCG immunoreactive cells were found in 5 squamous cell carcinomas (21 %) and in none of 5 non-squamous cell tumors. The hCG positive cells were found in the most infiltrating areas of the tumors where poorly differentiated and pleomorphic cells predominated. The positive tumors were 4 poorly differentiated (31 %) and one moderately differentiated carcinoma (12%). Four out of 10 cases (40%) with lymph node metastases had hCG in the primary tumor, whereas only one out of 11 cases (9%) without metastases was hCG positive. HPLand SP-l were found in two cases. These placental proteins were detected in similar areas than hCG but the number ofhPLand SP -1 immunoreactive cells was lower than hCG positive cells. SP-l was also seen in areas of squamous cell differentiation negative for hCG. None of these two cases showed trophoblastic differentiation.
Introduction Human chorionic gonadotropin (hCG) is a glycoprotein hormone produced by the normal placental syncytiotrophoblast and gestational and non-gestational trophoblastic tumors 2,25, 33. The hCG production in these situations is usually associated with the synthesis of other placental proteins such as human placental lactogen (hPL) and pregnancy-specific-beta-l glycoprotein (SP_l)I,19,25,27,28. Several studies have now demonstrated the presence of hCG and other placental proteins in visceral non-germ cell tumors 4,14,15,25. The biological significance of the placental protein production by non-trophoblastic neoplasm is not clear. In some cases, hCG has mainly been found in the most undifferentiated areas of the tumors 7,8, 18,23, and a relationship between their presence and a more aggressive behaviour of the tumor has been suggested I8 ,31. Human 0344-0338/91/0187-0044$3 .5010
placental lactogen and SP-l have been less assessed in visceral non-trophoblastic neoplasms. Gastro-intestinal adenocarcinomas have been shown to be associated with a high incidence of hCG production 6,7, 12, 13, 18. Esophageal carcinomas, as other squamous cell carcinomas, however, have not been so intensively studied. Serum studies have failed to demonstrate hCG in patients with esophageal carcinomas I4 ,15,25. Only three isolated cases of esophageal hCG producing tumors have been reported and none of them was a squamous cell carcinoma21 ,26,32. The aim of this study was to investigate the incidence of hCG production in esophageal carcinomas, to detect the expression of other placental proteins (hPL and SP-l) in hCG positive cases, and to correlate the presence of these trophoblastic markers with several clinicopathologic parameters.
©
1991
by Gustav Fischer Verlag, Stuttgart
HCG in Esophageal Carcinomas . 45
Material and Methods A total of 29 surgically removed esophageal tumors during a two-year period were examined. These included two women and 27 men ranging in age from 37 to 75 years (mean 45). The histology of the esophageal tumors were as follows: 2 adenoid cystic carcinomas, 2 adenocarcinomas, 1 adenosquamous carcinoma, and 24 squamous carcinomas. The latter were classified as well differentiated (3/24), moderately differentiated (8/24) and poorly differentiated (13/24) depending on their degree of keratinization and cellular atypia. Fresh specimens were submitted to our laboratory. They were opened and spread on a flat surface for fixation in 10% neutral formalin overnight. Representative samples were dehydrated with graded alcohols, rinsed in xylol and embedded in paraffin wax. SerialS [tm sections were cut and one of them was stained in each case with routine hematoxylin-eosin and for hCG immuno staining as a tissular marker for syncyiotrophoblast differentiation. The hCG positive cases were also immunohistochemically examined for other placental proteins such as hPL and SP-l, also detected in syncytiotrophoblast. Immunohistochemical staining was carried out using the avidin biotin complex technique (Vector]7). Primary rabbit antisera against beta-chain of hCG and hPL were obtained from Dakopatts (Copenhagen, Denmark), and antiserum against SP-l from Boehring (Marburg, West Germany). The antisera anti-hCG and anti-SP-l were used at a dilution of 11100 and the anti-serum anti-hPL at 1/200. The incubation with the primary anti-serum was performed at 4°C overnight followed by room temperature for one hour. Peroxidase activity was developed by a solution of 5 mg of 3,3'-diaminobenzidine tetrahydrochloride (DAB) (Sigma Chemical, St Louis MO) dissolved in 10 ml of 0.05 M tris buffer at pH 7.2 with 0.03% of HzO z. Normal trophoblast from placenta of different ages of gestation were used as control. These tissues were incubated with hCG, hLP and SP-l, obtaining a decoration of the syncytiotrophoblast. In other sections the primary antisera were replaced by PBS and by non-immune rabbit serum showing a negative staining of the syncytiotrophoblast.
Fig. 1. HCG immunoreactive cells (arrows) are predominant in the deeper areas of the tumor (immunoperoxidase-hematoxylin, X 100).
Positive results were classified as occasional, focal, and diffuse. Occasional tumors were those in which only some isolated positive cells were identified. Focal tumors were those in which clusters of positive cells were seen in some areas of the tumor but other regions were negative. Diffuse tumors were those in which isolated and/or clusters of positive cells were found throughout most areas of the tumor.
Results Tumor cells showing hCG immunoreactivity were found in five (21 %) of the 24 squamous cell carcinomas. No positivity was demonstrated in other histological types of esophageal carcinomas (Table 1). The hCG immunoreactive cells were usually found in the most infiltrating portion of the neoplasms where poorly differentiated and pleomorphic cells predominated (Fig. 1). The stained cells were seen as isolated or in small clusters infiltrating the stroma. The more keratinizing cells were negative. The immunostaining was localized in the cytoplasm of tumor cells with a diffuse pattern (Fig. 2). In one case, an
Table 1. HCG immunoreactivity in esophageal carcinomas Histologic Type
Total No
HCG+
Adenoid Cystic Adenocarcinoma Adenosquamous Squamous cell well differentiated moderately differentiad poorly differentiated
2 2 1 24
0 0 0 5
Total
29
3 8
0 1 4
13
5
46 . I. Trias et al. Table 2. Comparison of HCG-immunoreactivity in esophageal carcinomas and their local invasion Invasion of esophagic wall Total No
HCG+
PartiaP TotaJ2
2 3
12 12
1 Tumor limited to submucosa or muscularis propria. penetrated the full thickness of the wall.
2
Tumor
Table 3. Comparison of HCG immunoreactivity in esophageal carcinomas and the presence of lymph node metastases Presence of metastases
Patients!
HCG+
Negative Positive
11 10
1 4
1 Three cases could not be evaluated because of absence of lymph nodes in the surgical specimen.
Fig. 2. HCG immunoreactivity in the cytoplasm of pleomorphic cells with a diffuse pattern (immunoperoxidase-hematoxylin,
x 250).
additional focal paranuclear pattern was found in some cells (Fig. 3). The cells with this focal cytoplasmic pattern were diffusely distributed in small clusters throughout the tumor and showed a more differentiated appearance than the cells with a diffuse cytoplasmic pattern. The number of stained cells in the positive cases was variable. It was occasional in two, focal in one, and diffuse in two. No relationship could be found between the number of immunostained cells and the degree of differentiation, local invasiveness, or metastases. Tables 1, 2 and 3 show the relationships between the presence of hCG and the histologic classification of the tumors, their local invasion and involvement of regional lymph nodes. Four positive tumors were poorly differentiated carcinomas (31 %) and the other was a moderately differentiated one (12%). None of the well differentiated carcinomas was positive (Table 1). Four out of 10 (40%) cases with metastatic lymph nodes were hCG positive while only one out of 11 (9%) of the cases without metastasis had a positive immunostain (Table 3). Three cases could not be evaluated because of the absence of
Fig. 3. HCG immunoreactivity in clusters of squamous cells with a focal, paranuclear pattern (immunoperoxidase-hematoxylin,
x 250).
HCG in Esophageal Carcinomas· 47
Fig. 4. SP-l immunoreactivity in areas of keratinizing squamous cell carcinoma (immunoperoxidase-hematoxylin, x 150).
lymph nodes in the surgical specimen. No significant differences could be demonstrated between the presence of hCG and the degree of tumor invasiveness (Table 2). Human placental lactogen and SP-1 were detected in two cases. The histologic distribution of these proteins was similar to that of the hCG, but the number of hPL and SP-1 positive cells was less than for hCG in both cases. SP-1 reactivity was also found in areas of clear squamous differentiation where hCG was negative (Fig. 4). No clear trophoblastic differentiation could be seen in these two cases. They had, however, the most anaplastic cells seen in all five hCG positive cases (Fig. 2). Discussion In this study we have detected the presence of hCGimmunoreactive cells in 5 out of 24 esophageal squamous cell carcinomas (21 %). This is similar to that reported in gastric adenocarcinomas (11-51 %) 13.18,36, and in large bowel carcinomas (11-54%)6,7,12. Production of hCG by squamous cell carcinomas has also been detected in other
localizatiops.such as lung ll ,34, uterine cervix and vagina 9,IO. These findings and the detection of hCG in other types of carcinomas8,23 indicate that the production of this hormone by non-trophoblastic tumors is a frequent phenomenon not related to the histologic type of carcinomas. In our series 31 % (4 of 13) of the poorly differentiated squamous cell carcinomas were hCG positive whilst only 12% (1 of 8) of the moderately differentiated and none of the well differentiated were immunostained for this hormone. Positive cells were usually found in the less differentiated areas predominating in the infiltrating edge of tumors. HCG was also more frequently found in metastatic tumors (40%) than in neoplasms without lymph node metastasis (9%). Several studies in non-trophoblastic neoplasms have also found a relation between the presence of hCG and parameters of aggressive tumor behaviour such as high grade histologic type, greater local invasion, presence of metastases and resistance to treatment?, 8,10,18,20,23,31. The reasons for this association are not clear. HCG has been postulated to have immunosuppressive properties which would facilitate the progression of cellular clones producing the hormone 22 . However, the production of hCG by these tumors seems to be scarce since the number of positive cells is usually very low. Other authors have suggested that hCG may be produced by relatively undifferentiated stem cells responsible for cell renewal in the tissues 3,5. The immunohistochemical detection of hCG in tumors would indicate the presence of a greater number of proliferative undifferentiated cells. In this study we have observed two patterns of hCG immunoreactivity in the cytoplasm of neoplastic cells: focal paranuclear and diffuse. The focal paranuclear pattern was seen in clusters of neoplastic cells with a more differentiated appearance than those with the diffuse cytoplasmic pattern. A focal paranuclear reactivity has been previously detected for both alpha and beta hCG chains in several tumors 34 ,35. In a previous study in large bowel carcinomas?, we observed a diffuse cytoplasmic pattern in neoplastic glandular cells as well as in isolated undifferentiated cells whilst the focal paranuclear immunoreactivity was only seen in glandular differentiated cells. Our findings suggest that the cytoplasmic distribution of hCG immunoreactivity may be related with the degree of cell differentiation. The production of hCG by esophageal carcinomas was associated with the presence of hPL and SP-1 in two cases. Human placental lactogen and SP-1 have also been detected in other non-trophoblastic tumors 1,4, 25, 28 but the simultaneous presence of several placental proteins in the same tumor seems to be an infrequent phenomenon I6 ,30. Neither of these two cases had a clear choriocarcinomatous differentiation. To our knowledge, two esophageal tumors with morphologic trophoblastic differentiation have previously been published 21 ,32. One of them was a pure choriocarcinoma32 and, in the other, the trophoblastic elements were associated with an adenocarcinoma 21 . Sasano et aL26 reported an esophageal large cell ccarcinoma with areas resembling choriocarcinoma which produced high levels of hCG. These findings are similar to
48 . I. Trias et al.
other organs where the visceral heG producing tumors have been found to have three histologic patterns: pure choriocarcinomas, choriocarcinomas mixed with carcinoma, and carcinomas without trophoblastic elements 8,29. The relationship between all these histologic types is not clear. Morphological and immunohistochemical observations suggest that the choriocarcinomatous component in visceral tumors could progressively evolve from the carcinoma cells by a dedifferentiation process 8,24,29. The two cases observed in the present study which showed immunoreactivity for three placental proteins without choriocarcinomatous cells could represent an intermediate step in the process of trophoblastic differentiation.
References 1 Bagshave KD, Leguin RM, Sizaret PH, Tatarinov YS (1978) Pregnancy ~ 1 glycoprotein and chorionic gonadotropin in the serum of patients with trophoblastic and non-trophoblastic tumours. Eur J Cancer 14: 1331-1335 2 Bagshave KD (1983) Tumor markers: where do we go from here? Br J Cancer 48: 167-175 3 Baylin SB, Mendelshon G (1980) Ectopic (inappropiate) hormone production by tumors: Mechanisms involved and the histological and clinical implications. Endocr Rev 1: 45-77 4 Braunstein GA, VaitukaitisJL, Carbone PP, Ross GT (1973) Ectopic production of human chorionic gonaotropin by neoplasms. Ann Intern Med 78: 39-45 5 Braunstein GD, Rasor J, Wade ME (1980) Presence of and HCG-like substance in non-pregnant humans. In: Shegal ShJ (Ed) Chorionic Gonadotropin. Plenum Press, New York, 388-409 6 Buckley CH, Fox A (1979) An immunohistochemical study of the significance of HCG secretion by large bowel adenocarcinoma. J Clin Pathol 32: 368-372 7 Campo E, Palacfn A, Benasco C, Quesada E, Cardesa A (1987) Human chorionic gonadotropin in colorectal carcinoma. An immunohistochemical study. Cancer 59: 1611-1616 8 Campo E, Algaba F, Palacfn A, Germa R, Sole-Balcells FJ, Cardesa A (1989) Placental proteins in high grade urothelial neoplasms. An immunohistochemical study of human chorionic gonadotropin, human placental lactogen, and pregnancy-specific beta-I-glycoprotein. Cancer 63: 2497-2504 9 Das S, Mujkherjee K, Bhattacharya S, Chowdhury JR (1984) A comparative study of ectopic production of human chorionic gonadotropin and human chorionic somatotropin by carcinoma of the uterine cervix. Oncology 41: 303-307 10 Fenoglio CM, Hayata Y, Crum ChP, Richart RM (1982) The expression of human chorionic gonadotropin in the female genitale tract. Localization by the immuneperoxidase technique. Diag Gynecol Obstet 4: 97-103 11 Fukayama M, Hayashi Y, Koike M, Hajikano H, Endo S, Okumura H (1986) Human chorionic gonadotropin in lung and lung tumors. Immunohistochemical study of unbalanced distribution of subunits. Lab Invest 55: 433-442 12 Fukayama M, Hayashi Y, Koike M (1987a) Human chorionic gonadotropin in the rectosigmoid colon. Immunohistochemical study on unbalanced distribution of subunits. Am J Pathol 127: 83-89 13 Fukayama M, Hayashi Y, Koike M (1987b) Human chorionic gonadotropin in gastric carcinoma. An immunohistochemical study suggesting independent regulation of subunits. Virchows Arch A (pathol Anat) 411: 205-212
14 Gailani S, Chu TM, Hussbaum A, Ostrander M, ChristoffH (1976) Human chorionic gonadotropin in non-trophoblastic neoplasms. Assessment of abnormalities of HCG and CEA in bronchogenic and digestive neoplasms. Cancer 38: 1684-1686 15 Hattori M, Fukase M, Yoshimi H, Matsukura S, Imura H (1978) Ectopic production of human chorionic gonadotropin in malignant tumors. Cancer 42: 2328-2333 16 Heyderman E, Chapman DV, Richardson TC, Calvert I, Rosen SW (1985) Human chorionic gonadotropin and human placental lactogen in extragonadal tumors. An immunoperoxidase study of ten non-germ cell neoplasms. Cancer 56: 2674-2682 17 Hsu SM, Raine L, Fanger H (1981) Use of avidin-biotin peroxidase complex (ABC) in immunoperoxidase techniques: a comparison between ABC and PAP procedures. J. Histochem Cytochem 29: 557-580 18 Ito H, Tahara E (1983) Human chorionic gonadotropin in human gastric carcinoma. A retrospective immunohistochemical study. Acta Pathol Jpn 33: 287-296 19 Jacobsen GK, Jacobsen M, Clausen PP (1981) Distribution of tumor associated antigens in the various histologic components of germ cell tumors of the testis. Am J Surg Pathol 5: 257-266 20 Martin JE, Jenkis BJ, Zuk RJ, Oliver RTD, Baithum SI (1989) Human chorionic gonadotropin expression and histological findings as predictors of response to radiotherapy in carcinoma of the bladder. Virchows Archiv A (Pathol Anat) 414: 273-277 21 McKenchnie JC, Fechner RB (1971) Choriocarcinoma and adenocarcinoma of the esophagus with gonadotropin secretion. Cancer 26: 694-702 22 McManus LM, Naughton MA, Martinez-Hernandez A (1976) Human chorionic gonadotropin in human neoplastic cells. Cancer Res 36: 3476-3481 23 Nakamura Y, Unoura M, Noto H, Ohta G (1986) Human chorionic gonadotropin in primary liver carcinoma in adults. An immunohistochemical study. Virchows Arch A (Pathol Anat)
409: 365-373
24 Obe JA, Rosen N, Koss LG (1983) Primary choriocarcinoma of the urinary bladder. Report of a case with probable epithelial origin. Cancer 52: 1405-1409 25 Rosen SW, Weintraub BD, Vaitukaitis JL, Sussman HH, Hershanan JM, Muggia FM (1975) Placental proteins and their subunits as tumor markers. Ann Intern Med 82: 71-83 26 Sasano N, Abe S, Satake 0, Emdo K, Sato M (1970) Choriocarcinoma mimicry of an esophageal carcinoma with urinary gonadotropic activities. Tohoku J Exp Med 100: 153-163 27 Saxena BH, Goldstein AP, Emerson K, Selenkow HA (1968) Serum placental lactogen levels in patients with molar pregnancy and trophoblastic tumors. Am J Obstet Gynec 102: 115-121 28 Searle F, Bagshave KD, Leake BA, DentJ (1978) Serum SP-1 pregnancy specific ~-glycoprotein in choriocarcinoma and other neoplastic diseases Lancet 1: 579-581 29 Skrabanek P, KirraneJ, Powell D (1979) A unifying concept of chorionic gonadotropin production in malignancy. Invest Cell Pathol 2: 75-85 30 Sussman HH, Weintraub BD, Rosen SW (1974) Relationship of ectopic placental alkaline phosphatase to ectopic chorionic gonadotropin and placental lactogen: Discordance of three "markers" for cancer. Cancer 33: 820-823 31 Tormey DC, Waalkes TPh, Simon RM (1977) Biological markers in breast carcinoma II. Clinical correlations with human chorionic gonadotropin. Cancer 39: 2391-2396 32 Trillo AA, Accettullo LM, Yeiter TL (1979) Choriocarcinoma of the esophagus: Histologic and cytologic findings. A case report. Acta Cytol 23: 69-74
HCG in Esophageal Carcinomas . 49 33 Tyrey L (1982) Human chorionic gonadotropin: Structural, biologic, and immunologic aspects. Sem Oncol 9: 163-173 34 Wachner R, Wittekind Ch, von Kleist S (1984) Localization of CEA, ~-HCG, SP-l, and keratin in the tissue of lung carcinomas. An immunohistochemical study. Virchows Archiv A (Pathol Anat) 402: 415-423 35 Walker R (1978) Significance of a-subunit HCG demonstrated in breast carcinomas by the immunoperoxidase technique. J Clin Pathol 31: 245-249
36 Wittekind Ch, Wachner R, Henke W, von Kleist S (1986) Localization of CEA, HCG, lysozyme, alpha-I-antitrypsin and alpha-l-antichymotrypsin in gastric cancer and prognosis. Virchows Archiv A (Pathol Anat) 409: 715-724
Received January 23, 1990 . Accepted March 8, 1990
Key words: HCG - Esophageal carcinomas - Squamous cell carcinomas - Chorionic gonadotropin - Placental lactogen Antonio Palacin, MD, Departamento de Anatomia Patologica, Hospital Clinico y Provincial de Barcelona, Calle Villarroel 170, 08036-Barcelona, Spain
Human Chorionic Gonadotropin in Esophageal Carcinomas An Immunohistochemical Study I. Trias, E. Campo 1,2, C. Benasco, A. Palacin 1 and A. Cardesa Departments of Pathology, Hospital de Bellvitge "Princeps d'Espanya'; 1Hospital Clinico y Provincial de Barcelona, and 2Facultad de Medicina, Estudi General de Lleida, Universidad de Barcelona, Spain
SUMMARY We have examined immunohistochemically the presence of human chorionic gonadotrophin (hCG) in 29 esophageal carcinomas: 24 squamous cell carcinomas, 2 adenocarcinomas, 2 adenoid cystic carcinomas and 1 adenosquamous carcinoma. In hCG-positive tumors, the presence of human placental lactogen (hPL) and pregnancy-specific beta-l glycoprotein (SP-l) was also assessed. HCG immunoreactive cells were found in 5 squamous cell carcinomas (21 %) and in none of 5 non-squamous cell tumors. The hCG positive cells were found in the most infiltrating areas of the tumors where poorly differentiated and pleomorphic cells predominated. The positive tumors were 4 poorly differentiated (31 %) and one moderately differentiated carcinoma (12%). Four out of 10 cases (40%) with lymph node metastases had hCG in the primary tumor, whereas only one out of 11 cases (9%) without metastases was hCG positive. HPLand SP-l were found in two cases. These placental proteins were detected in similar areas than hCG but the number ofhPLand SP -1 immunoreactive cells was lower than hCG positive cells. SP-l was also seen in areas of squamous cell differentiation negative for hCG. None of these two cases showed trophoblastic differentiation.
Introduction Human chorionic gonadotropin (hCG) is a glycoprotein hormone produced by the normal placental syncytiotrophoblast and gestational and non-gestational trophoblastic tumors 2,25, 33. The hCG production in these situations is usually associated with the synthesis of other placental proteins such as human placental lactogen (hPL) and pregnancy-specific-beta-l glycoprotein (SP_l)I,19,25,27,28. Several studies have now demonstrated the presence of hCG and other placental proteins in visceral non-germ cell tumors 4,14,15,25. The biological significance of the placental protein production by non-trophoblastic neoplasm is not clear. In some cases, hCG has mainly been found in the most undifferentiated areas of the tumors 7,8, 18,23, and a relationship between their presence and a more aggressive behaviour of the tumor has been suggested I8 ,31. Human 0344-0338/91/0187-0044$3 .5010
placental lactogen and SP-l have been less assessed in visceral non-trophoblastic neoplasms. Gastro-intestinal adenocarcinomas have been shown to be associated with a high incidence of hCG production 6,7, 12, 13, 18. Esophageal carcinomas, as other squamous cell carcinomas, however, have not been so intensively studied. Serum studies have failed to demonstrate hCG in patients with esophageal carcinomas I4 ,15,25. Only three isolated cases of esophageal hCG producing tumors have been reported and none of them was a squamous cell carcinoma21 ,26,32. The aim of this study was to investigate the incidence of hCG production in esophageal carcinomas, to detect the expression of other placental proteins (hPL and SP-l) in hCG positive cases, and to correlate the presence of these trophoblastic markers with several clinicopathologic parameters.
©
1991
by Gustav Fischer Verlag, Stuttgart
HCG in Esophageal Carcinomas . 45
Material and Methods A total of 29 surgically removed esophageal tumors during a two-year period were examined. These included two women and 27 men ranging in age from 37 to 75 years (mean 45). The histology of the esophageal tumors were as follows: 2 adenoid cystic carcinomas, 2 adenocarcinomas, 1 adenosquamous carcinoma, and 24 squamous carcinomas. The latter were classified as well differentiated (3/24), moderately differentiated (8/24) and poorly differentiated (13/24) depending on their degree of keratinization and cellular atypia. Fresh specimens were submitted to our laboratory. They were opened and spread on a flat surface for fixation in 10% neutral formalin overnight. Representative samples were dehydrated with graded alcohols, rinsed in xylol and embedded in paraffin wax. SerialS [tm sections were cut and one of them was stained in each case with routine hematoxylin-eosin and for hCG immuno staining as a tissular marker for syncyiotrophoblast differentiation. The hCG positive cases were also immunohistochemically examined for other placental proteins such as hPL and SP-l, also detected in syncytiotrophoblast. Immunohistochemical staining was carried out using the avidin biotin complex technique (Vector]7). Primary rabbit antisera against beta-chain of hCG and hPL were obtained from Dakopatts (Copenhagen, Denmark), and antiserum against SP-l from Boehring (Marburg, West Germany). The antisera anti-hCG and anti-SP-l were used at a dilution of 11100 and the anti-serum anti-hPL at 1/200. The incubation with the primary anti-serum was performed at 4°C overnight followed by room temperature for one hour. Peroxidase activity was developed by a solution of 5 mg of 3,3'-diaminobenzidine tetrahydrochloride (DAB) (Sigma Chemical, St Louis MO) dissolved in 10 ml of 0.05 M tris buffer at pH 7.2 with 0.03% of HzO z. Normal trophoblast from placenta of different ages of gestation were used as control. These tissues were incubated with hCG, hLP and SP-l, obtaining a decoration of the syncytiotrophoblast. In other sections the primary antisera were replaced by PBS and by non-immune rabbit serum showing a negative staining of the syncytiotrophoblast.
Fig. 1. HCG immunoreactive cells (arrows) are predominant in the deeper areas of the tumor (immunoperoxidase-hematoxylin, X 100).
Positive results were classified as occasional, focal, and diffuse. Occasional tumors were those in which only some isolated positive cells were identified. Focal tumors were those in which clusters of positive cells were seen in some areas of the tumor but other regions were negative. Diffuse tumors were those in which isolated and/or clusters of positive cells were found throughout most areas of the tumor.
Results Tumor cells showing hCG immunoreactivity were found in five (21 %) of the 24 squamous cell carcinomas. No positivity was demonstrated in other histological types of esophageal carcinomas (Table 1). The hCG immunoreactive cells were usually found in the most infiltrating portion of the neoplasms where poorly differentiated and pleomorphic cells predominated (Fig. 1). The stained cells were seen as isolated or in small clusters infiltrating the stroma. The more keratinizing cells were negative. The immunostaining was localized in the cytoplasm of tumor cells with a diffuse pattern (Fig. 2). In one case, an
Table 1. HCG immunoreactivity in esophageal carcinomas Histologic Type
Total No
HCG+
Adenoid Cystic Adenocarcinoma Adenosquamous Squamous cell well differentiated moderately differentiad poorly differentiated
2 2 1 24
0 0 0 5
Total
29
3 8
0 1 4
13
5
46 . I. Trias et al. Table 2. Comparison of HCG-immunoreactivity in esophageal carcinomas and their local invasion Invasion of esophagic wall Total No
HCG+
PartiaP TotaJ2
2 3
12 12
1 Tumor limited to submucosa or muscularis propria. penetrated the full thickness of the wall.
2
Tumor
Table 3. Comparison of HCG immunoreactivity in esophageal carcinomas and the presence of lymph node metastases Presence of metastases
Patients!
HCG+
Negative Positive
11 10
1 4
1 Three cases could not be evaluated because of absence of lymph nodes in the surgical specimen.
Fig. 2. HCG immunoreactivity in the cytoplasm of pleomorphic cells with a diffuse pattern (immunoperoxidase-hematoxylin,
x 250).
additional focal paranuclear pattern was found in some cells (Fig. 3). The cells with this focal cytoplasmic pattern were diffusely distributed in small clusters throughout the tumor and showed a more differentiated appearance than the cells with a diffuse cytoplasmic pattern. The number of stained cells in the positive cases was variable. It was occasional in two, focal in one, and diffuse in two. No relationship could be found between the number of immunostained cells and the degree of differentiation, local invasiveness, or metastases. Tables 1, 2 and 3 show the relationships between the presence of hCG and the histologic classification of the tumors, their local invasion and involvement of regional lymph nodes. Four positive tumors were poorly differentiated carcinomas (31 %) and the other was a moderately differentiated one (12%). None of the well differentiated carcinomas was positive (Table 1). Four out of 10 (40%) cases with metastatic lymph nodes were hCG positive while only one out of 11 (9%) of the cases without metastasis had a positive immunostain (Table 3). Three cases could not be evaluated because of the absence of
Fig. 3. HCG immunoreactivity in clusters of squamous cells with a focal, paranuclear pattern (immunoperoxidase-hematoxylin,
x 250).
HCG in Esophageal Carcinomas· 47
Fig. 4. SP-l immunoreactivity in areas of keratinizing squamous cell carcinoma (immunoperoxidase-hematoxylin, x 150).
lymph nodes in the surgical specimen. No significant differences could be demonstrated between the presence of hCG and the degree of tumor invasiveness (Table 2). Human placental lactogen and SP-1 were detected in two cases. The histologic distribution of these proteins was similar to that of the hCG, but the number of hPL and SP-1 positive cells was less than for hCG in both cases. SP-1 reactivity was also found in areas of clear squamous differentiation where hCG was negative (Fig. 4). No clear trophoblastic differentiation could be seen in these two cases. They had, however, the most anaplastic cells seen in all five hCG positive cases (Fig. 2). Discussion In this study we have detected the presence of hCGimmunoreactive cells in 5 out of 24 esophageal squamous cell carcinomas (21 %). This is similar to that reported in gastric adenocarcinomas (11-51 %) 13.18,36, and in large bowel carcinomas (11-54%)6,7,12. Production of hCG by squamous cell carcinomas has also been detected in other
localizatiops.such as lung ll ,34, uterine cervix and vagina 9,IO. These findings and the detection of hCG in other types of carcinomas8,23 indicate that the production of this hormone by non-trophoblastic tumors is a frequent phenomenon not related to the histologic type of carcinomas. In our series 31 % (4 of 13) of the poorly differentiated squamous cell carcinomas were hCG positive whilst only 12% (1 of 8) of the moderately differentiated and none of the well differentiated were immunostained for this hormone. Positive cells were usually found in the less differentiated areas predominating in the infiltrating edge of tumors. HCG was also more frequently found in metastatic tumors (40%) than in neoplasms without lymph node metastasis (9%). Several studies in non-trophoblastic neoplasms have also found a relation between the presence of hCG and parameters of aggressive tumor behaviour such as high grade histologic type, greater local invasion, presence of metastases and resistance to treatment?, 8,10,18,20,23,31. The reasons for this association are not clear. HCG has been postulated to have immunosuppressive properties which would facilitate the progression of cellular clones producing the hormone 22 . However, the production of hCG by these tumors seems to be scarce since the number of positive cells is usually very low. Other authors have suggested that hCG may be produced by relatively undifferentiated stem cells responsible for cell renewal in the tissues 3,5. The immunohistochemical detection of hCG in tumors would indicate the presence of a greater number of proliferative undifferentiated cells. In this study we have observed two patterns of hCG immunoreactivity in the cytoplasm of neoplastic cells: focal paranuclear and diffuse. The focal paranuclear pattern was seen in clusters of neoplastic cells with a more differentiated appearance than those with the diffuse cytoplasmic pattern. A focal paranuclear reactivity has been previously detected for both alpha and beta hCG chains in several tumors 34 ,35. In a previous study in large bowel carcinomas?, we observed a diffuse cytoplasmic pattern in neoplastic glandular cells as well as in isolated undifferentiated cells whilst the focal paranuclear immunoreactivity was only seen in glandular differentiated cells. Our findings suggest that the cytoplasmic distribution of hCG immunoreactivity may be related with the degree of cell differentiation. The production of hCG by esophageal carcinomas was associated with the presence of hPL and SP-1 in two cases. Human placental lactogen and SP-1 have also been detected in other non-trophoblastic tumors 1,4, 25, 28 but the simultaneous presence of several placental proteins in the same tumor seems to be an infrequent phenomenon I6 ,30. Neither of these two cases had a clear choriocarcinomatous differentiation. To our knowledge, two esophageal tumors with morphologic trophoblastic differentiation have previously been published 21 ,32. One of them was a pure choriocarcinoma32 and, in the other, the trophoblastic elements were associated with an adenocarcinoma 21 . Sasano et aL26 reported an esophageal large cell ccarcinoma with areas resembling choriocarcinoma which produced high levels of hCG. These findings are similar to
48 . I. Trias et al.
other organs where the visceral heG producing tumors have been found to have three histologic patterns: pure choriocarcinomas, choriocarcinomas mixed with carcinoma, and carcinomas without trophoblastic elements 8,29. The relationship between all these histologic types is not clear. Morphological and immunohistochemical observations suggest that the choriocarcinomatous component in visceral tumors could progressively evolve from the carcinoma cells by a dedifferentiation process 8,24,29. The two cases observed in the present study which showed immunoreactivity for three placental proteins without choriocarcinomatous cells could represent an intermediate step in the process of trophoblastic differentiation.
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Received January 23, 1990 . Accepted March 8, 1990
Key words: HCG - Esophageal carcinomas - Squamous cell carcinomas - Chorionic gonadotropin - Placental lactogen Antonio Palacin, MD, Departamento de Anatomia Patologica, Hospital Clinico y Provincial de Barcelona, Calle Villarroel 170, 08036-Barcelona, Spain
