- Email: [email protected]
Expression of oncofoetal marker carcinoembryonic antigen in oral cancers in South India—a pilot study
Int. J. Oral Maxillofac. Surg. 2006; 35: 746–749 doi:10.1016/j.ijom.2006.02.012, available online at http://www.sciencedirect.com
Research Paper Head and Neck Oncology
Expression of oncofoetal marker carcinoembryonic antigen in oral cancers in South India—a pilot study
S. B. R. M. S. V. R. N.
Pai1,c, R. B. Pai1,a,c, Lalitha1,b, Kumaraswamy1, N. Lalitha1, Johnston2, M. K. Bhargava1
1 Kidwai Memorial Institute of Oncology, Hosur Road, Bangalore, India; 2Department of Biochemistry and Molecular Biology, The University of Calgary, Calgary, Alberta, Canada T2N 4N1
S. B. Pai, R. B. Pai, R. M. Lalitha, S. V. Kumaraswamy, N. Lalitha, R. N. Johnston, M. K. Bhargava: Expression of oncofoetal marker carcinoembryonic antigen in oral cancers in South India—a pilot study. Int. J. Oral Maxillofac. Surg. 2006; 35: 746– 749. # 2006 International Association of Oral and Maxillofacial Surgeons. Published by Elsevier Ltd. All rights reserved. Abstract. Expression of the oncofoetal glycoprotein, carcinoembryonic antigen (CEA), has been observed in a number of malignancies and is also being pursued as a target for anti-cancer therapy. This study explored the status of this biochemical entity in the oral squamous cell carcinoma (SCC) in South India caused by extensive chewing habits. Squamous cell carcinoma in the study belonged to grade I and grade II. Tumour staging of the patients recruited in the study ranged from T2N1M0 to T4N3M0. Of the grade II cases studied, 88% (7 out of 8) showed expression of CEA. The 2 cases of grade I SCC of buccal mucosa also showed positive anti-CEA staining. If the results from this pilot study can be validated with a larger sample size, a role can be attributed to this tumour marker in oral neoplasia, thereby opening up avenues for using CEA as an additional diagnostic marker in oral SCC in this population and as a possible target for anti-cancer therapy.
Expression of tumour-specific antigens such as the carcinoembryonic antigen (CEA) has been the hallmark of a number of neoplasms6. The presence or overexpression of CEA in a number of human cancers has provided researchers with the tools to characterize these tumours, as well as implement strategies to efficiently manage the cancer. CEA levels were reported to be elevated in the early stage of head and neck cancers11. Specific expression of the CEA has also been reported for tumours that originated from the salivary gland21. Other 0901-5027/080746 + 04 $30.00/0
forms of neoplasia such as oncolytic carcinoma15, as well as in aggressive and malignant odontogenic tumours8, show elevation in levels of the CEA oncofoetoprotein. Further significance for the identification and characterization of CEA as a biochemical marker lies in the possibility of using this glycoprotein as a diagnostic tool coupled with the other markers, as well as pathological analyses12. One example of such a scenario is the use of radiolabeled anti-CEA probe as an additional tool to characterize the tumours7. Recently, immu-
Key words: oral cancer; carcinoembryonic antigen; squamous cell carcinoma. Accepted for publication 8 February 2006
notherapeutic approaches to target cancer cells using vaccines against CEA have shown some promise experimentally either by stimulating cytotoxic T lymphocytic a Present address: Department of Pediatrics, Emory University School of Medicine, Atlanta, GA, USA (e-mail: pairb2000@ yahoo.com). b Present address: Department of Oral and Maxillofacial Surgery, M.S. Ramaiah Dental College and Hospital, Bangalore, India. c Contributed equally to the work.
# 2006 International Association of Oral and Maxillofacial Surgeons. Published by Elsevier Ltd. All rights reserved.
Carcinoembryonic antigen in oral cancers (CTL) response16 or by direct targeting of the cancer cells expressing CEA9. The presence of CEA in tumours is therefore gaining significance both from a diagnostic stand point and as an alternate target for cancer therapy. There are no reports of biochemical and molecular characterization of oral cancers from South Indian populations where this is a predominant form of cancer23. This class of tumours arises out of prolonged and constant insult of the buccal mucosa to agents in betel leaves, areca nut and tobacco4,5,18. Experimental studies with extracts of the components used in the betel quid have shown carcinogenic effect1. A better understanding of these malignancies would help the detailed study of the oncogenic process in these oral neoplasias, as well as making comparisons with tumours of other tissue origin. CEA is evolving in many cancers not only as a tumour-associated biochemical entity aiding in the diagnosis but also as an attractive target for vaccine-mediated therapy2. Studies on the expression of CEA in oral tumours, therefore, would allow the future exploration of the possibilities of CEA-mediated immunotherapy. This is significant in the light of the fact that a number of oral SCC fails to respond to conventional modalities of treatment. The present study attempts to evaluate the expression of CEA in a number of tumours of the oral cavity presented at the Kidwai Memorial Institute of Oncology, Bangalore, India. A representative fraction of the patients presenting with SCC of the buccal mucosa, presumably resulting from the chewing of smokeless tobacco for extended periods of time, were included in the current study. Patients and methods Patients and tumour samples
Patients presenting with tumours of the oral cavity at the Dental Department of the Kidwai Memorial Institute of Oncology, Bangalore, India were selected for the study. Ten patients with tumours in the various sites of the buccal cavity were enrolled in the study. The patients recruited in the current study were all habitual chewers of smokeless tobacco. The subjects leave the tobacco quid in the mouth for prolonged periods of time. The age of the patients ranged from 35 to 80 years. The SCC were diagnosed and graded by the pathologists. The criteria for grading as well as staging were as follows. Grade I: well differentiated; grade II: moderately differentiated. The following
parameters were used for tumour-nodemetastasis (TNM) staging. For tumour size: T1: 0–2 cm, T2: 2–4 cm, T3: >4 cm and T4: extensive spread of the tumour, or if there is involvement of nerve or bone; N0: no clinically palpable cervical lymph nodes or palpable nodes, but metastasis suspected, N1: clinically palpable-contra/homo lateral nodes that are not fixed, metastasis suspected, N2: clinically palpable contra lateral or bilateral cervical lymph nodes that are not fixed, metastasis suspected and N3: clinically palpable lymph nodes that are fixed, metastasis suspected; M0: no distant metastasis and M1: clinical and radiographic evidence of metastasis. Tissue specimens and immunohistochemistry
in the study were diagnosed as SCC, grade II. A large proportion of the tumours in the study were of T4 stage. Among the grade II SCC in the study, 88% showed moderate to strong staining to anti-CEA antibody. Only 1 case was negative for the presence of CEA. The 2 cases diagnosed as grade I SCC of the buccal cavity displayed abundant presence of CEA as observed by the strong immunoreactivity of anti-CEA antibody. A typical staining pattern of the tissues is shown in Fig. 1. Positive staining was observed in the sections from SCC grade II (Fig. 1A and C). Sections that were treated with all the components of the reaction except the antibody to CEA showed negative staining (Fig. 1B and D). Discussion
Tumour tissue samples were obtained by biopsy as well as by surgery. The tissue specimens were fixed in 10% formalin before embedding in paraffin wax. Sections of the tumour were stained with haematoxylin–eosin (H&E) and graded for the stage of the disease by the pathologists. For immunoreactivity studies with CEA, 5 mM paraffin sections on glass slides were deparaffinized and processed for studies. Antibodies to CEA were procured from Ortho Diagnostics, NJ, USA. Counterstaining was done with haematoxylin. Sections were mounted in glycerin for microscopic analysis. Treatment of tissue sections with all the reagents except the anti-CEA antibody served as the control. Analyses for the immunoreactivity of the antibodies were performed under light microscopy. Results
The results of the immunoreactivity studies conducted for the detection of CEA in the oral cancers are summarized in Table 1. The majority of cases enrolled
In the current pilot study the expression of CEA in the SCC of the oral buccal mucosa in the South Indian population has been examined. The studies show that in the oral oncogenic process induced by the insult due to chewing habits, CEA is expressed at an elevated level in grade I and the majority of the grade II SCC. Other reports have indicated an elevated level of CEA in the adenoid carcinoma from the maxillary sinus10 and 28% testing positive for CEA in head and neck cancers11. Serial estimations of CEA in the serum of patients harbouring tumours have been helpful in the follow-up of the patients22. Studies conducted on the upper aerodigestive cancers showed varying levels of expression of CEA24. In these tumours, expression of CEA was detected in all the adenocarcinoma. Similar to our observation with the SCC of the buccal mucosa, 73% of the SCC samples of the aerodigestive tract were positive for CEA. CEA, however, was not detected in basaloid-SCC as well as adenoid cystic carcinoma and the expression of this tumour
Table 1. Patient number
Age
Sex
TNM stage*
Histopathological details
1 2 3 4 5 6 7 8 9 10
45 80 35 40 70 40 45 45 70 40
F F F F F F F F F M
T4N3M0 T4N3M0 T2N1M0 T4N3M0 T3N3M0 T4N1M0 T4N1M0 T4N1M0 T4N1M0 T4N3M0
Ca. Ca. Ca. Ca. Ca. Ca. Ca. Ca. Ca. Ca.
*
747
buccal buccal buccal buccal buccal buccal buccal buccal buccal buccal
mucosa mucosa mucosa mucosa mucosa mucosa mucosa mucosa mucosa mucosa
SCC, SCC, SCC, SCC, SCC, SCC, SCC, SCC, SCC, SCC,
Staining for CEAy grade grade grade grade grade grade grade grade grade grade
II II I II II I II II II II
+++ +++ ++ + +++ ++ ++ ++ +++
The criteria employed for TNM staging is detailed in the Patients and Methods section. (+++) intense positive staining; (++) moderate staining; (+) weak positive staining; () negative staining. y
748
Pai et al.
Fig. 1. Immunohistochemical staining of oral squamous cell carcinoma (SCC) with anti-CEA antibody: deparaffinized sections from 2-patient samples with SCC grade II were stained with anti-CEA antibody as mentioned in Patients and Methods. (A and C) Positive staining in the tumour cells. (B and D) Sections from the respective tumour tissues processed without incorporation of the anti-CEA antibody, showing negative staining (original magnification 40).
marker occurred at a low frequency in mucoepidermoid carcinoma (29%). This suggests the differential evolution of the various lineages of carcinoma as well as a probable role for CEA in the SCC of the upper aero-digestive tract. KASS et al. reported a high percentage of head and neck cancers to be positive for CEA in their study9. In a similar study, 40% of the oral tumours were positive for CEA in comparison to 78% of the tumours of maxillary origin25. Advanced oral dysplastic lesions also show a concurrent increase in CEA levels19. For more differentiated SCC, CEA was found to be a useful marker17. The detection of the presence of CEA in the oral SCC adds an additional tool to the repertoire of pathologists diagnosing tumours. The observations from this pilot study need to be validated with recruitment of patients encompassing a full spectrum of tumour stages and grades along with appropriate control tissues. It is becoming increasingly evident that a combination of markers would be a powerful screening tool for oral cancers13. In the oral cancers studied here, the presence of the CEA oncofoetoprotein opens up ave-
nues for the intervention of these tumours by immunotherapy protocols. Research in this direction has already shown promise with respect to using CEA-mediated immunotherapy for colon cancers through the MHC class I and class II molecules. The lack of toxicity of some of the CEAmediated vaccine therapy has prompted researchers to introduce this modality in clinical trials (for a review of literature see SAROBE et al.20). Experimental studies with CEA-expressing cells from head and neck cancers have demonstrated that induction of CEA-mediated cytotoxic T lymphocytic response may be feasible in these cancer cells to inhibit their growth9. Several other gene therapy approaches also exploit the unique expression of CEA in a number of cancers2,3,14. More detailed studies with larger sample size are needed to determine the possible utility of CEA as a diagnostic tool in oral SCC from this region. Information gathered from extensive studies combined with the ones presented in this manuscript should allow the determination of the utility of this marker as an anticancer therapy target, especially when current modalities of chemotherapy, radiation and surgery are not effective.
The genesis of oral cancer in the population studied, caused by the physical insult by the quid as well as the chemical constituents therein, represents a unique pathway of conversion of the normal buccal mucosa to a neoplastically transformed state. Additional studies with larger sample size and in conjunction with other tumour-specific markers should provide insights into the oral carcinogenic process as well as aid in the management of this disease, which causes high morbidity and mortality. Acknowledgements. The study was supported by the Council of Scientific and Industrial Research, New Delhi, India and Indian Council of Medical Research, New Delhi, India.
References 1. Bhide SV, Shah AS, Nair J, Nagarajrao D. Epidemiological and experimental studies on tobacco-related oral cancer in India. IARC Sci Publ 1984: 851–857. 2. Brinkman JA, Fausch SC, Weber JS, Kast WM. Peptide-based vaccines for
Carcinoembryonic antigen in oral cancers
3.
4.
5.
6.
7. 8.
9.
10.
cancer immunotherapy. Expert Opin Biol Ther 2004: 4: 181–198. Chung-Faye GA, Kerr DJ, Young LS, Searle PF. Gene therapy strategies for colon cancer. Mol Med Today 2000: 6: 82–87. Dayal PK, Mani NJ, Bhargava K. Prevalence of oral cancer and precancerous lesions in ‘pan’/‘supari’ chewers. Indian J Public Health 1978: 22: 234– 245. Gupta PC, Mehta FS, Daftary DK, Pindborg JJ, Bhonsle RB, Jalnawalla PN, Sinor PN, Pitkar VK, Murti PR, Irani RR, Shah HT, Kadam PM, Iyer KS, Iyer HM, Hegde AK, Chandrashekar GK, Shiroff BC, Sahiar BE, Mehta MN. Incidence rates of oral cancer and natural history of oral precancerous lesions in a 10-year followup study of Indian villagers. Community Dent Oral Epidemiol 1980: 8: 283– 333. Hammarstrom S. The carcinoembryonic antigen (CEA) family: structures, suggested functions and expression in normal and malignant tissues. Semin Cancer Biol 1999: 9: 67–81. Hopsu EV, Kairemo KJ. Radioimmunodetection of head and neck cancer. A review. Acta Oncol 1993: 32: 735–740. Howell RE, Handlers JP, Aberle AM, Abrams AM, Melrose RJ. CEA immunoreactivity in odontogenic tumors and keratocysts. Oral Surg Oral Med Oral Pathol 1988: 66: 576–580. Kass ES, Greiner JW, Kantor JA, Tsang KY, Guadagni F, Chen Z, Clark B, De Pascalis R, Schlom J, Van Waes C. Carcinoembryonic antigen as a target for specific antitumor immunotherapy of head and neck cancer. Cancer Res 2002: 62: 5049–5057. Kishimoto H, Urade M, Sakurai K, Noguchi K. Isolation and characterisation of adenoid squamous carcinoma cells highly producing SCC antigen and CEA
11.
12.
13.
14.
15.
16.
17.
18. 19.
from carcinoma of the maxillary sinus. Oral Oncol 2000: 36: 70–75. Kuo WR, Lee KW, Ho KY, Tsai SM, Chiang FY, Juan KH. Tissue polypeptide antigen, carcinoembryonic antigen, carbohydrate antigen, and CA125 levels as tumor markers in squamous cell carcinoma of the head and neck. Kaohsiung J Med Sci 1999: 15: 152–158. Kurokawa H, Tsuru S, Okada M, Nakamura T, Kajiyama M. Evaluation of tumor markers in patients with squamous cell carcinoma in the oral cavity. Int J Oral Maxillofac Surg 1993: 22: 35–38. Kurokawa H, Yamashita Y, Tokudome S, Kajiyama M. Combination assay for tumor markers in oral squamous cell carcinoma. J Oral Maxillofac Surg 1997: 55: 964–966. Li Y, Chen Y, Dilley J, Arroyo T, Ko D, Working P, Yu DC. Carcinoembryonic antigen-producing cell-specific oncolytic adenovirus, OV798, for colorectal cancer therapy. Mol Cancer Ther 2003: 2: 1003–1009. Muramatsu T, Hashimoto S, Lee MW, Chung CK, Matsuzaki K, Inoue T, Noma H, Shimono M. Oncocytic carcinoma arising in submandibular gland with immunohistochemical observations and review of the literature. Oral Oncol 2003: 39: 199–203. Parsons T, Spendlove I, Nirula R, Writer M, Carter G, Carr F, Durrant LG. A novel CEA vaccine stimulates T cell proliferation, gammaIFN secretion and CEA specific CTL responses. Vaccine 2004: 22: 3487–3494. Rosati G, Riccardi F, Tucci A. Use of tumor markers in the management of head and neck cancer. Int J Biol Markers 2000: 15: 179–183. Sanghvi L, Rao DDN, Joshi S. Epidemiology of head and neck cancers. Semin Surg Oncol 1989: 5: 305–309. Sano K. Morphometric and immunohistochemical investigation of oral epithelial
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23. 24.
25.
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dysplasia and squamous cell carcinoma. Shika Kiso Igakkai Zasshi 1989: 31: 436– 452. Sarobe P, Huarte E, Lasarte JJ, Borras-Cuesta F. Carcinoembryonic antigen as a target to induce anti-tumor immune responses. Curr Cancer Drug Targets 2004: 4: 443–454. Seifert G, Caselitz J. Markers of oral and salivary gland tumors: immunocytochemical investigations. Cancer Detect Prev 1985: 8: 23–34. Silverman NA, Alexander Jr JC, Chretien PB. CEA levels in head and neck cancer. Cancer 1976: 37: 2204– 2211. Sturgis EM. A review of social and behavioral efforts at oral cancer preventions in India. Head Neck 2004: 26: 937–944. Tsubochi H, Suzuki T, Suzuki S, Ohashi Y, Ishibashi S, Moriya T, Fujimura S, Sasano H. Immunohistochemical study of basaloid squamous cell carcinoma, adenoid cystic and mucoepidermoid carcinoma in the upper aerodigestive tract. Anticancer Res 2000: 20: 1205–1211. Yanagawa T, Hayashi Y, Nishida T, Yoshida H, Yura Y, Azuma M, Sato M. Immunohistochemical demonstration of carcinoembryonic antigen (CEA) on tissue sections from squamous cell head and neck cancer and plasma CEA levels of the patients. Int J Oral Maxillofac Surg 1986: 15: 296–306.
Address: Rekha B. Pai Veterans Affairs Medical Center Medical Research 151H 1670 Clairmont Road Decatur GA 30033 USA Tel: +1 404 321 6111x2272 Fax: +1 404 728 7726 E-mail: [email protected]
Research Paper Head and Neck Oncology
Expression of oncofoetal marker carcinoembryonic antigen in oral cancers in South India—a pilot study
S. B. R. M. S. V. R. N.
Pai1,c, R. B. Pai1,a,c, Lalitha1,b, Kumaraswamy1, N. Lalitha1, Johnston2, M. K. Bhargava1
1 Kidwai Memorial Institute of Oncology, Hosur Road, Bangalore, India; 2Department of Biochemistry and Molecular Biology, The University of Calgary, Calgary, Alberta, Canada T2N 4N1
S. B. Pai, R. B. Pai, R. M. Lalitha, S. V. Kumaraswamy, N. Lalitha, R. N. Johnston, M. K. Bhargava: Expression of oncofoetal marker carcinoembryonic antigen in oral cancers in South India—a pilot study. Int. J. Oral Maxillofac. Surg. 2006; 35: 746– 749. # 2006 International Association of Oral and Maxillofacial Surgeons. Published by Elsevier Ltd. All rights reserved. Abstract. Expression of the oncofoetal glycoprotein, carcinoembryonic antigen (CEA), has been observed in a number of malignancies and is also being pursued as a target for anti-cancer therapy. This study explored the status of this biochemical entity in the oral squamous cell carcinoma (SCC) in South India caused by extensive chewing habits. Squamous cell carcinoma in the study belonged to grade I and grade II. Tumour staging of the patients recruited in the study ranged from T2N1M0 to T4N3M0. Of the grade II cases studied, 88% (7 out of 8) showed expression of CEA. The 2 cases of grade I SCC of buccal mucosa also showed positive anti-CEA staining. If the results from this pilot study can be validated with a larger sample size, a role can be attributed to this tumour marker in oral neoplasia, thereby opening up avenues for using CEA as an additional diagnostic marker in oral SCC in this population and as a possible target for anti-cancer therapy.
Expression of tumour-specific antigens such as the carcinoembryonic antigen (CEA) has been the hallmark of a number of neoplasms6. The presence or overexpression of CEA in a number of human cancers has provided researchers with the tools to characterize these tumours, as well as implement strategies to efficiently manage the cancer. CEA levels were reported to be elevated in the early stage of head and neck cancers11. Specific expression of the CEA has also been reported for tumours that originated from the salivary gland21. Other 0901-5027/080746 + 04 $30.00/0
forms of neoplasia such as oncolytic carcinoma15, as well as in aggressive and malignant odontogenic tumours8, show elevation in levels of the CEA oncofoetoprotein. Further significance for the identification and characterization of CEA as a biochemical marker lies in the possibility of using this glycoprotein as a diagnostic tool coupled with the other markers, as well as pathological analyses12. One example of such a scenario is the use of radiolabeled anti-CEA probe as an additional tool to characterize the tumours7. Recently, immu-
Key words: oral cancer; carcinoembryonic antigen; squamous cell carcinoma. Accepted for publication 8 February 2006
notherapeutic approaches to target cancer cells using vaccines against CEA have shown some promise experimentally either by stimulating cytotoxic T lymphocytic a Present address: Department of Pediatrics, Emory University School of Medicine, Atlanta, GA, USA (e-mail: pairb2000@ yahoo.com). b Present address: Department of Oral and Maxillofacial Surgery, M.S. Ramaiah Dental College and Hospital, Bangalore, India. c Contributed equally to the work.
# 2006 International Association of Oral and Maxillofacial Surgeons. Published by Elsevier Ltd. All rights reserved.
Carcinoembryonic antigen in oral cancers (CTL) response16 or by direct targeting of the cancer cells expressing CEA9. The presence of CEA in tumours is therefore gaining significance both from a diagnostic stand point and as an alternate target for cancer therapy. There are no reports of biochemical and molecular characterization of oral cancers from South Indian populations where this is a predominant form of cancer23. This class of tumours arises out of prolonged and constant insult of the buccal mucosa to agents in betel leaves, areca nut and tobacco4,5,18. Experimental studies with extracts of the components used in the betel quid have shown carcinogenic effect1. A better understanding of these malignancies would help the detailed study of the oncogenic process in these oral neoplasias, as well as making comparisons with tumours of other tissue origin. CEA is evolving in many cancers not only as a tumour-associated biochemical entity aiding in the diagnosis but also as an attractive target for vaccine-mediated therapy2. Studies on the expression of CEA in oral tumours, therefore, would allow the future exploration of the possibilities of CEA-mediated immunotherapy. This is significant in the light of the fact that a number of oral SCC fails to respond to conventional modalities of treatment. The present study attempts to evaluate the expression of CEA in a number of tumours of the oral cavity presented at the Kidwai Memorial Institute of Oncology, Bangalore, India. A representative fraction of the patients presenting with SCC of the buccal mucosa, presumably resulting from the chewing of smokeless tobacco for extended periods of time, were included in the current study. Patients and methods Patients and tumour samples
Patients presenting with tumours of the oral cavity at the Dental Department of the Kidwai Memorial Institute of Oncology, Bangalore, India were selected for the study. Ten patients with tumours in the various sites of the buccal cavity were enrolled in the study. The patients recruited in the current study were all habitual chewers of smokeless tobacco. The subjects leave the tobacco quid in the mouth for prolonged periods of time. The age of the patients ranged from 35 to 80 years. The SCC were diagnosed and graded by the pathologists. The criteria for grading as well as staging were as follows. Grade I: well differentiated; grade II: moderately differentiated. The following
parameters were used for tumour-nodemetastasis (TNM) staging. For tumour size: T1: 0–2 cm, T2: 2–4 cm, T3: >4 cm and T4: extensive spread of the tumour, or if there is involvement of nerve or bone; N0: no clinically palpable cervical lymph nodes or palpable nodes, but metastasis suspected, N1: clinically palpable-contra/homo lateral nodes that are not fixed, metastasis suspected, N2: clinically palpable contra lateral or bilateral cervical lymph nodes that are not fixed, metastasis suspected and N3: clinically palpable lymph nodes that are fixed, metastasis suspected; M0: no distant metastasis and M1: clinical and radiographic evidence of metastasis. Tissue specimens and immunohistochemistry
in the study were diagnosed as SCC, grade II. A large proportion of the tumours in the study were of T4 stage. Among the grade II SCC in the study, 88% showed moderate to strong staining to anti-CEA antibody. Only 1 case was negative for the presence of CEA. The 2 cases diagnosed as grade I SCC of the buccal cavity displayed abundant presence of CEA as observed by the strong immunoreactivity of anti-CEA antibody. A typical staining pattern of the tissues is shown in Fig. 1. Positive staining was observed in the sections from SCC grade II (Fig. 1A and C). Sections that were treated with all the components of the reaction except the antibody to CEA showed negative staining (Fig. 1B and D). Discussion
Tumour tissue samples were obtained by biopsy as well as by surgery. The tissue specimens were fixed in 10% formalin before embedding in paraffin wax. Sections of the tumour were stained with haematoxylin–eosin (H&E) and graded for the stage of the disease by the pathologists. For immunoreactivity studies with CEA, 5 mM paraffin sections on glass slides were deparaffinized and processed for studies. Antibodies to CEA were procured from Ortho Diagnostics, NJ, USA. Counterstaining was done with haematoxylin. Sections were mounted in glycerin for microscopic analysis. Treatment of tissue sections with all the reagents except the anti-CEA antibody served as the control. Analyses for the immunoreactivity of the antibodies were performed under light microscopy. Results
The results of the immunoreactivity studies conducted for the detection of CEA in the oral cancers are summarized in Table 1. The majority of cases enrolled
In the current pilot study the expression of CEA in the SCC of the oral buccal mucosa in the South Indian population has been examined. The studies show that in the oral oncogenic process induced by the insult due to chewing habits, CEA is expressed at an elevated level in grade I and the majority of the grade II SCC. Other reports have indicated an elevated level of CEA in the adenoid carcinoma from the maxillary sinus10 and 28% testing positive for CEA in head and neck cancers11. Serial estimations of CEA in the serum of patients harbouring tumours have been helpful in the follow-up of the patients22. Studies conducted on the upper aerodigestive cancers showed varying levels of expression of CEA24. In these tumours, expression of CEA was detected in all the adenocarcinoma. Similar to our observation with the SCC of the buccal mucosa, 73% of the SCC samples of the aerodigestive tract were positive for CEA. CEA, however, was not detected in basaloid-SCC as well as adenoid cystic carcinoma and the expression of this tumour
Table 1. Patient number
Age
Sex
TNM stage*
Histopathological details
1 2 3 4 5 6 7 8 9 10
45 80 35 40 70 40 45 45 70 40
F F F F F F F F F M
T4N3M0 T4N3M0 T2N1M0 T4N3M0 T3N3M0 T4N1M0 T4N1M0 T4N1M0 T4N1M0 T4N3M0
Ca. Ca. Ca. Ca. Ca. Ca. Ca. Ca. Ca. Ca.
*
747
buccal buccal buccal buccal buccal buccal buccal buccal buccal buccal
mucosa mucosa mucosa mucosa mucosa mucosa mucosa mucosa mucosa mucosa
SCC, SCC, SCC, SCC, SCC, SCC, SCC, SCC, SCC, SCC,
Staining for CEAy grade grade grade grade grade grade grade grade grade grade
II II I II II I II II II II
+++ +++ ++ + +++ ++ ++ ++ +++
The criteria employed for TNM staging is detailed in the Patients and Methods section. (+++) intense positive staining; (++) moderate staining; (+) weak positive staining; () negative staining. y
748
Pai et al.
Fig. 1. Immunohistochemical staining of oral squamous cell carcinoma (SCC) with anti-CEA antibody: deparaffinized sections from 2-patient samples with SCC grade II were stained with anti-CEA antibody as mentioned in Patients and Methods. (A and C) Positive staining in the tumour cells. (B and D) Sections from the respective tumour tissues processed without incorporation of the anti-CEA antibody, showing negative staining (original magnification 40).
marker occurred at a low frequency in mucoepidermoid carcinoma (29%). This suggests the differential evolution of the various lineages of carcinoma as well as a probable role for CEA in the SCC of the upper aero-digestive tract. KASS et al. reported a high percentage of head and neck cancers to be positive for CEA in their study9. In a similar study, 40% of the oral tumours were positive for CEA in comparison to 78% of the tumours of maxillary origin25. Advanced oral dysplastic lesions also show a concurrent increase in CEA levels19. For more differentiated SCC, CEA was found to be a useful marker17. The detection of the presence of CEA in the oral SCC adds an additional tool to the repertoire of pathologists diagnosing tumours. The observations from this pilot study need to be validated with recruitment of patients encompassing a full spectrum of tumour stages and grades along with appropriate control tissues. It is becoming increasingly evident that a combination of markers would be a powerful screening tool for oral cancers13. In the oral cancers studied here, the presence of the CEA oncofoetoprotein opens up ave-
nues for the intervention of these tumours by immunotherapy protocols. Research in this direction has already shown promise with respect to using CEA-mediated immunotherapy for colon cancers through the MHC class I and class II molecules. The lack of toxicity of some of the CEAmediated vaccine therapy has prompted researchers to introduce this modality in clinical trials (for a review of literature see SAROBE et al.20). Experimental studies with CEA-expressing cells from head and neck cancers have demonstrated that induction of CEA-mediated cytotoxic T lymphocytic response may be feasible in these cancer cells to inhibit their growth9. Several other gene therapy approaches also exploit the unique expression of CEA in a number of cancers2,3,14. More detailed studies with larger sample size are needed to determine the possible utility of CEA as a diagnostic tool in oral SCC from this region. Information gathered from extensive studies combined with the ones presented in this manuscript should allow the determination of the utility of this marker as an anticancer therapy target, especially when current modalities of chemotherapy, radiation and surgery are not effective.
The genesis of oral cancer in the population studied, caused by the physical insult by the quid as well as the chemical constituents therein, represents a unique pathway of conversion of the normal buccal mucosa to a neoplastically transformed state. Additional studies with larger sample size and in conjunction with other tumour-specific markers should provide insights into the oral carcinogenic process as well as aid in the management of this disease, which causes high morbidity and mortality. Acknowledgements. The study was supported by the Council of Scientific and Industrial Research, New Delhi, India and Indian Council of Medical Research, New Delhi, India.
References 1. Bhide SV, Shah AS, Nair J, Nagarajrao D. Epidemiological and experimental studies on tobacco-related oral cancer in India. IARC Sci Publ 1984: 851–857. 2. Brinkman JA, Fausch SC, Weber JS, Kast WM. Peptide-based vaccines for
Carcinoembryonic antigen in oral cancers
3.
4.
5.
6.
7. 8.
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