Carcinoembryonic Antigen (CEA) in the Gastric Mucosa after Partial Gastrectomy K. G. JANUNGER, J. LINDGREN, P. SIPPONEN & L. DOMELLOF Dept. of Surgery, University Hospital, Umei, Sweden, Dept. of Bacteriology and Immunology, University of Helsinki, Helsinki, and Dept. of Pathology, Jorvi Hospital, Espoo, Finland
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Janunger. K. G.. Lindpen, J., Sipponen. P. & Domellof, L. Carcinoembryonic antigen (CEA) in the gastric mucosa after partial gastrectomy. Scund. J. Gustroenl. 1979, 1 4 , 5 5 5-560. Carcinoembryonic antigen (CEA) was studied by the three-layer bridge immunoperoxidase technique in gastric biopsy specimens taken from 49 patients, 13-20 years after partial gastrectomy. F.outine histological examination revealed various degrees of chronic atrophic gastritis in all patients. A positive CEA reaction was found in 6 out of 9 with malignant or premalignant mucosal changes and in 4 out of 40 without these changes. In two cases of carcinoma the biopsies revealed a positive CEA reaction. In 4 patients with carinoma diagnosed 1-2 years after the first examination the initial nonmalignant biopsies wen: CEA-positive in one case. All biopsies from mucosa with severe dysplasia and adenomatous polyps were CEA-positive. Four patients without malignant or premalignant changes in the gastric mucosa had CEA-positive biopsies. No carcinoma has been found in these patients at re-examinations after 1 year. The results indicate that the occurrence of imniunohistochemically detectable CEA may be associated with malignant transformation of the gastric mucosa The value of this method in screening patients at increased risk of gastric carcinoma will be further explored.
Key-words: Carcinoem bryogenic antigen; duodenal ulcer; endoscopy; gastrectomy ; gastric cancer; gastric polyps; gastric ulcer L . DomelloL M.D., Depl. of Surgery. Universi1.y Hospital, S-901 85 Umea. Sweden
Several histological changes in the gastric mucosa have been discussed in terms of pre-malignancy: chronic atrophic gastritis (27, 32), intestinal metaplasia (14, 16, 21, 24), cystic dilatation of the gastric glands (22), and adenomatous polyps (19, 30). These changes seem to be more frequent in the gastric remnant after partial gastrectomy than in the non-operated stomach (6-8). An increased prevalence of gastric-stump carcinoma has been reported by several authors (8, 9, 11, 15, 20), but it has not been possible to ascertain a correlation between the histological changes and an increased risk for carcinoma in the operated stomach (7). Normal gastric mucosa has not been found to contain carcinoembryonic antigen (CEA) when studied by immunohistochemical methods (3, 26), but in gastric carcinoma this is a common finding (4, 5, 26). The aim of the present investigation was to study the occurrence of CEA in the mucosa of the
gastric remnant of such patients with or without severe dysplasia, polyps, or cancer, to evaluate whether this immunohistochemical method can be used as an early indicator of gastric malignancy. MATERIAL AND METHODS Forty-nine patients who had been submitted to partial gastrectomy for benign ulcer disease 13-20 years earlier were offered repeated follow-ups with gastroscopy and a minimum of six biopsies, as previously described (6,7). These patients belong to a major follow-up study started in 1973. During this survey biopsies for histochemical analyses were taken from carcinomas in 2 patients, from nonmalignant mucosa in 4 patients in whom carcinoma was diagnosed 1-2 years later, from 2 patients with severe dysplasia in the biopsies, and from polyps in 19 patients (1 adenoma. 18 hyperplastic polyps).
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K. G. Janunger, J. Lindgren, P. Sipponen & L . Domellof
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In addition, biopsies from 22 patients were studied, of whom 1 1 had intestinal metaplasia demonstrated in the biopsy specimens. All the patients had histologically proven chronic atrophic gastritis. Forty-six patients were operated on according to Billroth I1 and three according to Billroth I. For comparison, we studied biopsies with histologically normal gastric mucosa taken from five healthy patients without gastric disease. Serum CEA determination In all patients, serum CEA was determined by radioimmunoassay as described by Stenman et al. (28). The cut-off level was 5.0 ng/ml (25). In three patients with gastric carcinoma these determinations were made after radical total gastrectomy. CEA and antiserum to CEA CEA was purified from liver metastases of colon cancer using procedures described previously ( 10). The homogenized tissue was extracted with 1 M perchloric acid (PCA), centrifuged at 10,000 g for 15 min, and the supernatant was lyophilized. The final purification consisted of two gel filtrations, on Sepharose 4 B and Sephadex G-200 columns, respectively, and atlinity chromatography on a concanavalin A-Sepharose column (Pharmacia, Uppsala, Sweden). Antiserum to CEA was produced in rabbits. Immunization was performed with 200500 pg of CEA in complete Freund's adjuvant given subcutaneously and intramuscularly at 2-week intervals. The animals were bled 1 week after the third injection. Thereafter booster injections were given and the animals were bled at monthly intervals. The antiserum was absorbed with Sepharose-coupled PCA extract of normal human spleen, normal gastric juice, meconium, and with normal human A, B, and 0 erythrocytes. After this procedure the antiserum showed the following immunohistochemical and immunological characteristics: 1) it stained the brush border of colonic cancer tissue, 2) it occasionally stained the brush border of normal colonic mucosa but did not stain goblet cells, 3) these staining properties could be abolished by absorption of the serum with CEA,
4) it did not stain neutrophils in formalin-fixed
specimens, 5) in immunodiffusion it precipitated purified CEA but formed no precipitate with normal human serum or with PCA extracts of normal spleen and gastric juice. A weak precipitate was formed with PCA extract of meconium, but this precipitation line showed complete identity with CEA, and 6) it did not agglutinate A, B, or 0 erythrocytes. Other antisera Rabbit IgG was prepared by 50% ammonium sulphate precipitation followed by DEAE-cellulose ion-exchange chromatography. Antiserum to this antigen was produced in sheep. Anti-peroxidase serum was prepared in a rabbit by using purified horseradish peroxidase (Sigma, Type VI) as the antigen. The immunization schedule was the same as for antiserum to CEA. CEA staining CEA was stained in formalin-fixed, deparafbized histological sections by using the three-layer bridge immunoperoxidase method of Primus et al. (23). Briefly, sections were first treated with rabbit antiserum to CEA diluted 1/30, followed by sheep antiserum to rabbit IgG and rabbit antiserum to peroxidase, in dilutions of 1/50 and 1/ 100, respectively. Finally, sections were treated with peroxidase (horseradish peroxidase, Sigma, Type VI, 1 mg/ 10 ml). and the enzyme reaction was developed for 5 min in 0.05 M Tris buffer, pH 7.6, containing 0.075% 3,3'-diaminobenzidine and 0.05% H,O,. Prior to staining of CEA, the endogenous peroxidase activity was destroyed by incubation of dep a r a n i z e d sections for 15 min in methanol containing 0.5% of H,O,. All dilutions and washings before and after each step in the staining procedure were made in phosphate-buffered saline (PBS) containing 2% normal sheep serum. Control sections were stained with antiserum to CEA absorbed with CEA. Sections were lightly counterstained with haematoxylin. RESULTS A positive immunohistochemical reaction with the antiserum to CEA was found in the mucosal biopsies from 10 out of 49 operated patients, but in none
CEA afier Partial Gastrectomy Table I. C E A reactivity in malignant and non-malignant mucosal biopsies
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Total Diffuse-type carcinoma Nonmalignant mucosa in patients with later carcinoma Severe dysplasia Adenomatous polyp Hyperplastic polyps Atrophic gastritis with intestinal metaplasia Atrophic gastritis without intestinal metaplasia Total Histologically normal gastric mucosa (healthy controls)
2
Positive C EA reaction 2
4 2 1 18
II
2
II 49
10
5
-
I
of the biopsies from the antral or body mucosa of 5 healthy, non-operated control patients (Table I). CEA was found in biopsies taken from two histologically verified stump carcinomas. Both were of the diffuse type according to the histological classilication of Laurtn ( 17). In these carcinomas the C EA reactivity was localized to the cytoplasm of mitlignant cells (Fig. 1) but varied in intensity from cell to cell. Of four patients with carcinoma diagnosed 1-2 years after the first examination the initial nonmalignant biopsies were CEA-positive in one case. In this patient, operated on with a subtotal gastrec-
551
tomy, the positive CEA reaction could be demonstrated in mucosal biopsies pre-operatively and at a post-operative re-examination. Biopsies from two patients with severe cellular and structural dysplasia (pre-cancerous changes) showed positive CEA reaction. One of them has been subjected to a gastric reresection. The surgical specimen was cut in 20-30 thin slices, and 3 sections of each slice was examined histologically. This examination confirmed the presence of severe dysplasia, but no malignancy could be found. The other patient has not been reoperated. CEA was also found in histological sections from one adenomatous polyp extirpated by cauterization at the gastroscopy. In the remaining 40 patients CEA was found in biopsies from 1 patient with hyperplastic polyp, in 2 patients with intestinal metaplasia, and in 1 patient without this alteration. CEA was seen in the luminal border of the foveolar epithelial cells (Fig. 2), whereas cells deeper in the gastric glands were regularly negative. CEA was not present in the metaplastic epithelium. Intracellular CEA was not seen. In patients with positive CEA staining the individual biopsies were compared for the presence of CEA. No patient revealed positive CEA reactivity in all the six biopsies taken. Elevated serum CEA levels were not found in any of the patients. However, in three patients with stump carcinoma, serum CEA was examined only after gastric re-resection.
Fig. 1. CEA reaction in malignant cells of diffuse carcinoma of the gastric stump. Lightly counterstained with haemaloxylin ( ~ 5 5 0 ) .
558
K . G. Janunger, J . Lindgren. P. Sipponen
C?
L . Domellof
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a
Fig. 2a. A typical CEA reaction in the luminal border of foveolar epithelial cells in nonmalignant stump mucosa. Lightly counterstained with haematoxylin ( ~400).
Fig. 2b. A control section stained with anti-CEA antiserum absorbed with purified CEA. Note the absence of positive reaction in the luminal border of the foveolar epithelium when compared with Fig. 2a. Lightly counterstained with haematoxylin
(x400).
DISCUSSION CEA has not been found immunohistochemically in normal adult gastric mucosa, but it has been demonstrated in gastric carcinoma irrespective of the histological type (1, 18, 26) and in the surrounding mucosa with intestinal metaplasia (2, 5 ) . The occurrence of CEA reactivity has not been studied in patients with polyps or severe dysplasia or in
patients at high risk of developing gastric carcinoma-for example those with pernicious anaemia, chronic atrophic gastritis, or previous partial gastrectomy. In this preliminary study a positive CEA reaction was found in six out of nine patients with histologically demonstrated pre-malignant or malignant gastric mucosa. Three of the patients with CEA-negative mucosa later developed a gastric-stump
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CEA afler Partial Gastrectom-v
carcinoma. A possible explanation of the negative CEA reactivity in these cases may be that C13A seems to have a patchy distribution in the mucosa.. It is also reported that CEA is not demonstrable in all gastric carcinomas when highly specific antisera are used (3-5). Antisera that react with CEA and CEiArelated antigens show a higher frequency of CE:Apositive gastric carcinomas ( 2 6 ) , but part of this staining is probably due to reactions with crossreacting antigens such as NCA, NCA,, or CELJA (3, 13, 31). The positive mucosal CEA staining in the patients with severe dysplasia and adenoma indicates that the occurrence of CEA in gastric mucosa may coincide with the malignant transformation. Three Billroth-11-operated patients with positive CEA reactions but without polyps or marked clysplasia were re-examined after 2 years. The macroand micro-scopic findings were unchanged. In these patients the presence of CEA was not found togel her with intestinal metaplasia. This is in accordance with recent findings reported by Burtin et al. (3), who demonstrated that the previously observed positive reaction in intestinal metaplasia was mainly due to the presence of NCA,. These findings are interesting since intestinal metaplasia has repeatedly been reported as a pre-malignant condition. The validity of an immunohistochemical study depends on the specificity of the antiserum used. To achieve specificity, our antiserum to CEA was carefully absorbed. Cross-reactions with NCA ( 13) and CEA-like antigen in normal gastric juice, CELIA (31), were excluded by absorption with PCA extracts of normal spleen and normal gastric juice. To avoid cross-reaction with NCA, (2, 3), our antiserum to CEA was absorbed with PCA extract of meconium. After this procedure the serum gave a precipitate against meconium showing immunological identity with CEA, but no spur formation was observed (25). In addition, our serum showed immunohistochemical staining characteristics typical of CEA (3, 12). Hence we consider that CEA-like antigens did not interfere with out immunoperoxidase analyses. The immunohistochemical detection of CEA may contribute to the identification of some patients at risk of developing gastric carcinoma. However, at present the sensitivity and specificity of this method are not sufficient for clinical use. The
559
possibility of improving the antisera will be further studied. ACKNOWLEDGEMENTS This work was supported by grants from the Finnish Cancer Society, The Finnish Cultural Foundation, and Swedish Cancer Society project no. 797-B7704XC. REFERENCES I. Bordes, M.. Michiels, R. & Martin, F. Digestion 1973, 9, 106-115 2. Burtin, P.. Hirsch-Marie, H. & Chavanel, G. J. Immunol. 1973, 6 , 1926-1928 3. Burtin, P., Sabine, M. C. & Chavanel. G. Int. J. Cancer 1977, 19, 634-641 4. Denk, H., Tappeiner, G., Davidovits, A., Eckerstorfer. R. & Holzner, J. H. J. Natl. Cancer Inst. 1974, 53, 933-942 5. Denk, H., Tappeiner, G., Davidovits, A. & Holzner. J. H. Virchows Arch. Abt. A 1973, 360, 339-347 6. Domellof, L., Eriksson, S . & Janunger, K. G. Amer. J. Surg. 1976. 132, 26-31 7. Domellof, L., Eriksson, S . & Janunger, K. G. Gastroenterology 1977, 73, 462-468 8. Domellof. L. & Janunger, K. G. Amer. J. Surg. 1977, 134, 581-584 9. Griesser, G. & Schmidt, H. Med. Welt ( B e d . ) 1964, 35, 1936-1940 10. Hammarstrom, S . , Engvall, E. & Sundblad, G. in Bostrom, H. & Larsson, T. (eds.) Health Control in Detection of Cancer. Almqvist & Wicksell, Internat., Stockholm, 1976 1 I. Helsingen, N. & Hillestad, L. Ann. Surg. 1956, 143, 173- 179 12. Huitric, E. pp. 469-472 in Peeters, H. (ed.) Protides of the Biological Fluids. Pergamon Press, Oxford, New York. 1976 13. von Kleist, S., Chavanel, G. & Burtin, P. Proc. Natl. Acad. Sci. (USA) 1972, 69, 2492-2494 14. Korn. E. R. Amer. J. Gastroent. 1974, 61, 27&275 15. Krause, U. Acta Chir. Scand. 1958, 114, 341-354 16. Krentz, K. Acta Hepatogastroent. 1973, 20, 226235 17. Lauren, P. Acta Path. Microbiol. Scand. 1965, 64, 3 1-49 18. Martin, F. & Martin, M. S . Int. J. Cancer. 1972, 9, 64 1-647 19. Ming, S. C. & Goldman, H. Cancer 1965, 18, 721726 20. Morgenstern, L., Yamakawa, T. & Seltzer, D. Amer. J. S&g. 1973, 125, 29-38 21. Morson, B. C. Brit. J. Cancer 1955, 9. 365-376 22. Nagayo, T. Acta Path. Jap. 1974, 24, (2), 249-272 23. Primus, J. F., Wang, R. H., Sharkey, R.M. & Goldenberg, D. M. J . Immunol Meth. 1975, 8, 267-276 24. Reynolds, K. W., Johnson, A. G. & Fox, B. Clin. Oncol. 1975, I , 101-109
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25. Rutanen, E. M., Lindgren, J., Sipponen, P., Stenman, tides of the Biological Fluids. Pergamon Press, OxU. H., Saksela, E. S. & Seppala, M. Cancer 1978,42, ford, New York, 1976 58 1-590 29. Thomson, D. M. P., Krupey, J. & Freedman, S. 0. 26. Sipponen, P., Ruoslahti. E., Vuento, M., Engvall, E., Proc. Natl. Acad. Sci. ( U S A ) 1969. 64. 161-167 Stenman, U., Ihamaki, T. & Siurala, M. Acta Hepato- 30. Tomasulo, J. Cancer 1971. 27. 1346-1355 gastroent. 1916, 23. 276-219 31. Vuento, M., Ruoslahti, E., Pihko, H., Svenberg. T.. 27. Siurala, M., Lehtola, J. & Ihamaki. T. Scand. J. Ihamaki, T. & Siurala, M. Immunochemistrv 1976. Gastroent. 1974, 9. 441-446 13, 313-316 28. Stenman, U. H., Seppala, M.. Rutanen, E. M. & 32. Walker, I. R., Strickland. R. G., Ungar. B. & Mackay. Ruoslahti, E. pp. 457-460 in Peeters, H. (ed.) ProI. R. Gut 1971, 12, 906-91 1
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Received 9 December 1978 Accepted 20 February 1979
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Janunger. K. G.. Lindpen, J., Sipponen. P. & Domellof, L. Carcinoembryonic antigen (CEA) in the gastric mucosa after partial gastrectomy. Scund. J. Gustroenl. 1979, 1 4 , 5 5 5-560. Carcinoembryonic antigen (CEA) was studied by the three-layer bridge immunoperoxidase technique in gastric biopsy specimens taken from 49 patients, 13-20 years after partial gastrectomy. F.outine histological examination revealed various degrees of chronic atrophic gastritis in all patients. A positive CEA reaction was found in 6 out of 9 with malignant or premalignant mucosal changes and in 4 out of 40 without these changes. In two cases of carcinoma the biopsies revealed a positive CEA reaction. In 4 patients with carinoma diagnosed 1-2 years after the first examination the initial nonmalignant biopsies wen: CEA-positive in one case. All biopsies from mucosa with severe dysplasia and adenomatous polyps were CEA-positive. Four patients without malignant or premalignant changes in the gastric mucosa had CEA-positive biopsies. No carcinoma has been found in these patients at re-examinations after 1 year. The results indicate that the occurrence of imniunohistochemically detectable CEA may be associated with malignant transformation of the gastric mucosa The value of this method in screening patients at increased risk of gastric carcinoma will be further explored.
Key-words: Carcinoem bryogenic antigen; duodenal ulcer; endoscopy; gastrectomy ; gastric cancer; gastric polyps; gastric ulcer L . DomelloL M.D., Depl. of Surgery. Universi1.y Hospital, S-901 85 Umea. Sweden
Several histological changes in the gastric mucosa have been discussed in terms of pre-malignancy: chronic atrophic gastritis (27, 32), intestinal metaplasia (14, 16, 21, 24), cystic dilatation of the gastric glands (22), and adenomatous polyps (19, 30). These changes seem to be more frequent in the gastric remnant after partial gastrectomy than in the non-operated stomach (6-8). An increased prevalence of gastric-stump carcinoma has been reported by several authors (8, 9, 11, 15, 20), but it has not been possible to ascertain a correlation between the histological changes and an increased risk for carcinoma in the operated stomach (7). Normal gastric mucosa has not been found to contain carcinoembryonic antigen (CEA) when studied by immunohistochemical methods (3, 26), but in gastric carcinoma this is a common finding (4, 5, 26). The aim of the present investigation was to study the occurrence of CEA in the mucosa of the
gastric remnant of such patients with or without severe dysplasia, polyps, or cancer, to evaluate whether this immunohistochemical method can be used as an early indicator of gastric malignancy. MATERIAL AND METHODS Forty-nine patients who had been submitted to partial gastrectomy for benign ulcer disease 13-20 years earlier were offered repeated follow-ups with gastroscopy and a minimum of six biopsies, as previously described (6,7). These patients belong to a major follow-up study started in 1973. During this survey biopsies for histochemical analyses were taken from carcinomas in 2 patients, from nonmalignant mucosa in 4 patients in whom carcinoma was diagnosed 1-2 years later, from 2 patients with severe dysplasia in the biopsies, and from polyps in 19 patients (1 adenoma. 18 hyperplastic polyps).
556
K. G. Janunger, J. Lindgren, P. Sipponen & L . Domellof
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In addition, biopsies from 22 patients were studied, of whom 1 1 had intestinal metaplasia demonstrated in the biopsy specimens. All the patients had histologically proven chronic atrophic gastritis. Forty-six patients were operated on according to Billroth I1 and three according to Billroth I. For comparison, we studied biopsies with histologically normal gastric mucosa taken from five healthy patients without gastric disease. Serum CEA determination In all patients, serum CEA was determined by radioimmunoassay as described by Stenman et al. (28). The cut-off level was 5.0 ng/ml (25). In three patients with gastric carcinoma these determinations were made after radical total gastrectomy. CEA and antiserum to CEA CEA was purified from liver metastases of colon cancer using procedures described previously ( 10). The homogenized tissue was extracted with 1 M perchloric acid (PCA), centrifuged at 10,000 g for 15 min, and the supernatant was lyophilized. The final purification consisted of two gel filtrations, on Sepharose 4 B and Sephadex G-200 columns, respectively, and atlinity chromatography on a concanavalin A-Sepharose column (Pharmacia, Uppsala, Sweden). Antiserum to CEA was produced in rabbits. Immunization was performed with 200500 pg of CEA in complete Freund's adjuvant given subcutaneously and intramuscularly at 2-week intervals. The animals were bled 1 week after the third injection. Thereafter booster injections were given and the animals were bled at monthly intervals. The antiserum was absorbed with Sepharose-coupled PCA extract of normal human spleen, normal gastric juice, meconium, and with normal human A, B, and 0 erythrocytes. After this procedure the antiserum showed the following immunohistochemical and immunological characteristics: 1) it stained the brush border of colonic cancer tissue, 2) it occasionally stained the brush border of normal colonic mucosa but did not stain goblet cells, 3) these staining properties could be abolished by absorption of the serum with CEA,
4) it did not stain neutrophils in formalin-fixed
specimens, 5) in immunodiffusion it precipitated purified CEA but formed no precipitate with normal human serum or with PCA extracts of normal spleen and gastric juice. A weak precipitate was formed with PCA extract of meconium, but this precipitation line showed complete identity with CEA, and 6) it did not agglutinate A, B, or 0 erythrocytes. Other antisera Rabbit IgG was prepared by 50% ammonium sulphate precipitation followed by DEAE-cellulose ion-exchange chromatography. Antiserum to this antigen was produced in sheep. Anti-peroxidase serum was prepared in a rabbit by using purified horseradish peroxidase (Sigma, Type VI) as the antigen. The immunization schedule was the same as for antiserum to CEA. CEA staining CEA was stained in formalin-fixed, deparafbized histological sections by using the three-layer bridge immunoperoxidase method of Primus et al. (23). Briefly, sections were first treated with rabbit antiserum to CEA diluted 1/30, followed by sheep antiserum to rabbit IgG and rabbit antiserum to peroxidase, in dilutions of 1/50 and 1/ 100, respectively. Finally, sections were treated with peroxidase (horseradish peroxidase, Sigma, Type VI, 1 mg/ 10 ml). and the enzyme reaction was developed for 5 min in 0.05 M Tris buffer, pH 7.6, containing 0.075% 3,3'-diaminobenzidine and 0.05% H,O,. Prior to staining of CEA, the endogenous peroxidase activity was destroyed by incubation of dep a r a n i z e d sections for 15 min in methanol containing 0.5% of H,O,. All dilutions and washings before and after each step in the staining procedure were made in phosphate-buffered saline (PBS) containing 2% normal sheep serum. Control sections were stained with antiserum to CEA absorbed with CEA. Sections were lightly counterstained with haematoxylin. RESULTS A positive immunohistochemical reaction with the antiserum to CEA was found in the mucosal biopsies from 10 out of 49 operated patients, but in none
CEA afier Partial Gastrectomy Table I. C E A reactivity in malignant and non-malignant mucosal biopsies
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Total Diffuse-type carcinoma Nonmalignant mucosa in patients with later carcinoma Severe dysplasia Adenomatous polyp Hyperplastic polyps Atrophic gastritis with intestinal metaplasia Atrophic gastritis without intestinal metaplasia Total Histologically normal gastric mucosa (healthy controls)
2
Positive C EA reaction 2
4 2 1 18
II
2
II 49
10
5
-
I
of the biopsies from the antral or body mucosa of 5 healthy, non-operated control patients (Table I). CEA was found in biopsies taken from two histologically verified stump carcinomas. Both were of the diffuse type according to the histological classilication of Laurtn ( 17). In these carcinomas the C EA reactivity was localized to the cytoplasm of mitlignant cells (Fig. 1) but varied in intensity from cell to cell. Of four patients with carcinoma diagnosed 1-2 years after the first examination the initial nonmalignant biopsies were CEA-positive in one case. In this patient, operated on with a subtotal gastrec-
551
tomy, the positive CEA reaction could be demonstrated in mucosal biopsies pre-operatively and at a post-operative re-examination. Biopsies from two patients with severe cellular and structural dysplasia (pre-cancerous changes) showed positive CEA reaction. One of them has been subjected to a gastric reresection. The surgical specimen was cut in 20-30 thin slices, and 3 sections of each slice was examined histologically. This examination confirmed the presence of severe dysplasia, but no malignancy could be found. The other patient has not been reoperated. CEA was also found in histological sections from one adenomatous polyp extirpated by cauterization at the gastroscopy. In the remaining 40 patients CEA was found in biopsies from 1 patient with hyperplastic polyp, in 2 patients with intestinal metaplasia, and in 1 patient without this alteration. CEA was seen in the luminal border of the foveolar epithelial cells (Fig. 2), whereas cells deeper in the gastric glands were regularly negative. CEA was not present in the metaplastic epithelium. Intracellular CEA was not seen. In patients with positive CEA staining the individual biopsies were compared for the presence of CEA. No patient revealed positive CEA reactivity in all the six biopsies taken. Elevated serum CEA levels were not found in any of the patients. However, in three patients with stump carcinoma, serum CEA was examined only after gastric re-resection.
Fig. 1. CEA reaction in malignant cells of diffuse carcinoma of the gastric stump. Lightly counterstained with haemaloxylin ( ~ 5 5 0 ) .
558
K . G. Janunger, J . Lindgren. P. Sipponen
C?
L . Domellof
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a
Fig. 2a. A typical CEA reaction in the luminal border of foveolar epithelial cells in nonmalignant stump mucosa. Lightly counterstained with haematoxylin ( ~400).
Fig. 2b. A control section stained with anti-CEA antiserum absorbed with purified CEA. Note the absence of positive reaction in the luminal border of the foveolar epithelium when compared with Fig. 2a. Lightly counterstained with haematoxylin
(x400).
DISCUSSION CEA has not been found immunohistochemically in normal adult gastric mucosa, but it has been demonstrated in gastric carcinoma irrespective of the histological type (1, 18, 26) and in the surrounding mucosa with intestinal metaplasia (2, 5 ) . The occurrence of CEA reactivity has not been studied in patients with polyps or severe dysplasia or in
patients at high risk of developing gastric carcinoma-for example those with pernicious anaemia, chronic atrophic gastritis, or previous partial gastrectomy. In this preliminary study a positive CEA reaction was found in six out of nine patients with histologically demonstrated pre-malignant or malignant gastric mucosa. Three of the patients with CEA-negative mucosa later developed a gastric-stump
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CEA afler Partial Gastrectom-v
carcinoma. A possible explanation of the negative CEA reactivity in these cases may be that C13A seems to have a patchy distribution in the mucosa.. It is also reported that CEA is not demonstrable in all gastric carcinomas when highly specific antisera are used (3-5). Antisera that react with CEA and CEiArelated antigens show a higher frequency of CE:Apositive gastric carcinomas ( 2 6 ) , but part of this staining is probably due to reactions with crossreacting antigens such as NCA, NCA,, or CELJA (3, 13, 31). The positive mucosal CEA staining in the patients with severe dysplasia and adenoma indicates that the occurrence of CEA in gastric mucosa may coincide with the malignant transformation. Three Billroth-11-operated patients with positive CEA reactions but without polyps or marked clysplasia were re-examined after 2 years. The macroand micro-scopic findings were unchanged. In these patients the presence of CEA was not found togel her with intestinal metaplasia. This is in accordance with recent findings reported by Burtin et al. (3), who demonstrated that the previously observed positive reaction in intestinal metaplasia was mainly due to the presence of NCA,. These findings are interesting since intestinal metaplasia has repeatedly been reported as a pre-malignant condition. The validity of an immunohistochemical study depends on the specificity of the antiserum used. To achieve specificity, our antiserum to CEA was carefully absorbed. Cross-reactions with NCA ( 13) and CEA-like antigen in normal gastric juice, CELIA (31), were excluded by absorption with PCA extracts of normal spleen and normal gastric juice. To avoid cross-reaction with NCA, (2, 3), our antiserum to CEA was absorbed with PCA extract of meconium. After this procedure the serum gave a precipitate against meconium showing immunological identity with CEA, but no spur formation was observed (25). In addition, our serum showed immunohistochemical staining characteristics typical of CEA (3, 12). Hence we consider that CEA-like antigens did not interfere with out immunoperoxidase analyses. The immunohistochemical detection of CEA may contribute to the identification of some patients at risk of developing gastric carcinoma. However, at present the sensitivity and specificity of this method are not sufficient for clinical use. The
559
possibility of improving the antisera will be further studied. ACKNOWLEDGEMENTS This work was supported by grants from the Finnish Cancer Society, The Finnish Cultural Foundation, and Swedish Cancer Society project no. 797-B7704XC. REFERENCES I. Bordes, M.. Michiels, R. & Martin, F. Digestion 1973, 9, 106-115 2. Burtin, P.. Hirsch-Marie, H. & Chavanel, G. J. Immunol. 1973, 6 , 1926-1928 3. Burtin, P., Sabine, M. C. & Chavanel. G. Int. J. Cancer 1977, 19, 634-641 4. Denk, H., Tappeiner, G., Davidovits, A., Eckerstorfer. R. & Holzner, J. H. J. Natl. Cancer Inst. 1974, 53, 933-942 5. Denk, H., Tappeiner, G., Davidovits, A. & Holzner. J. H. Virchows Arch. Abt. A 1973, 360, 339-347 6. Domellof, L., Eriksson, S . & Janunger, K. G. Amer. J. Surg. 1976. 132, 26-31 7. Domellof, L., Eriksson, S . & Janunger, K. G. Gastroenterology 1977, 73, 462-468 8. Domellof. L. & Janunger, K. G. Amer. J. Surg. 1977, 134, 581-584 9. Griesser, G. & Schmidt, H. Med. Welt ( B e d . ) 1964, 35, 1936-1940 10. Hammarstrom, S . , Engvall, E. & Sundblad, G. in Bostrom, H. & Larsson, T. (eds.) Health Control in Detection of Cancer. Almqvist & Wicksell, Internat., Stockholm, 1976 1 I. Helsingen, N. & Hillestad, L. Ann. Surg. 1956, 143, 173- 179 12. Huitric, E. pp. 469-472 in Peeters, H. (ed.) Protides of the Biological Fluids. Pergamon Press, Oxford, New York. 1976 13. von Kleist, S., Chavanel, G. & Burtin, P. Proc. Natl. Acad. Sci. (USA) 1972, 69, 2492-2494 14. Korn. E. R. Amer. J. Gastroent. 1974, 61, 27&275 15. Krause, U. Acta Chir. Scand. 1958, 114, 341-354 16. Krentz, K. Acta Hepatogastroent. 1973, 20, 226235 17. Lauren, P. Acta Path. Microbiol. Scand. 1965, 64, 3 1-49 18. Martin, F. & Martin, M. S . Int. J. Cancer. 1972, 9, 64 1-647 19. Ming, S. C. & Goldman, H. Cancer 1965, 18, 721726 20. Morgenstern, L., Yamakawa, T. & Seltzer, D. Amer. J. S&g. 1973, 125, 29-38 21. Morson, B. C. Brit. J. Cancer 1955, 9. 365-376 22. Nagayo, T. Acta Path. Jap. 1974, 24, (2), 249-272 23. Primus, J. F., Wang, R. H., Sharkey, R.M. & Goldenberg, D. M. J . Immunol Meth. 1975, 8, 267-276 24. Reynolds, K. W., Johnson, A. G. & Fox, B. Clin. Oncol. 1975, I , 101-109
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Received 9 December 1978 Accepted 20 February 1979
