- Email: [email protected]
Gamma-glutamyl transpeptidase activity in human oral squamous cell carcinoma
Gamma-glutamyl transpeptidase activity in human oral squamous cell carcinoma D. Mock,* B. Whitestone,** MOUNT
SINAI
and J. Freeman,***
Toronto, Ontario, Canada
HOSPITAL
Gamma-glutamyl transpeptidase (GGT) activity has been shown to increase in preneoplastic cells in various experimental carcinogenesis models and in some human tumors. This pilot study was undertaken to examine its potential use in detection of sites of human oral carcinogenesis. Samples were taken from 16 surgical specimens: 12 resection specimens for squamous carcinomas and 4 benign mucosal lesions. Twelve of the patients were tobacco users. Diffuse GGT activity was found in all tobacco users and heavy cellular activity in all carcinomas. In four of the carcinoma-resection specimens, occasional cells staining heavily for GGT activity were noted in epithelium distant from the tumor itself. These results are discussed in relation to the potential value of GGT activity as a marker from preneoplastic changes in oral epithelium. (ORAL SURG. ORAL MED. ORAL PATHOL. 1987;64:197-201)
Gamma-glutamyl transpeptidase (GGT) is a membrane-bound enzyme found in epithelial cells of jejunal villi, pancreatic acini, bile ducts, seminal vesicles, the choroid plexus, and proximal convoluted tubules of kidney,’ as well as in lymphocytes.* This glycoprotein catalyzes the transfer of gamma-glutamy1 moiety of peptides to other peptides and amino acids. Therefore, the physiologic role of GGT is thought to be related to translocation of amino acids across cell membranes.3 GGT has proved useful as a histochemical marker for preneoplastic cells in the rat liver:5 in experimentally induced hepatomas,6*7and in neoplastic mouse skin.* Limited studies have reported increased GGT activity in some human cancers.9*LoIn untreated hamster cheek pouch mucosa and in hamster pouch mucosa treated with noncarcinogenic irritants, GGT activity was not evident,11-‘3In epithelial whole mounts of DMBA-exposed hamster cheek pouch mucosa, GGT was detected histochemically in localized areas of dysplasia and in well-differentiated squamous cell carcinomas.‘4vIs These preliminary studies suggest that GGT may be useful as a marker in oral squamous cell carcinomas and precancerous lesions.
*Department of Pathology and Dentistry. **Department of Dentistry. ***Department of Otolaryngology.
MATERIALS AND METHODS Tissue samples
Mucosal samples were obtained from 16 patients undergoing diagnostic or therapeutic surgical procedures of the oral cavity. Included were only those patients for whom adequate documentation of smoking, alcohol intake, previous related biopsies, and relevant clinical history was available. Multiple samples were taken, including both neoplastic tissue and adjacent grossly unremarkable tissue, from the tissue resection specimens. Tissue samples were obtained immediately after resection of the relevant area. The tissueswere quick-frozen, and cryostat-cut sections 5 pm in thickness were obtained. Histochemistry
The cryostat-cut sections of fresh frozen tissue were mounted on glass slides. Histochemical localization of GGT activity was carried out according to the method of Rutenburg and coworkers.L6 The sections were incubated for 30 minutes at room temperature in fresh medium containing gammaglutamyl-Cmethoxy 2 naphthylamide, fast blue BB salt, and glycylglycine. After rinsing with 0.85% sodium chloride, the tissues were dipped in 0.2 mol/L cupric sulfate solution, rinsed again in 0.85% sodium chloride, and washed in distilled water. The sections were counterstained with hematoxylin. Slides were coverslipped with glycerin gelatin mounting medium. Sections of kidney, rich in GGT 197
198
Mock, Whitestone, and Freeman
Oral Surg. August 1987
Table I GGT staining Patient No.
Age/Sex
Tobacco use
Alcohol
use
25/M 12/F 26/M 45/F 54/F
None None Cigarette Cigarette None
Social None Social Social Moderate
70/F
Cigarette
Moderate
7
50/F
Cigarette
8
77/M
Cigarette
Heavy
9
66/M
Cigarette
Moderate
10
56/M
Cigarette
Heavy
11
83/F
None
Social
12
65/F
Cigarette
Heavy
13
39/F
Cigarette
Moderate
14
37/M
Cigarette
Heavy
15 16
58/M 59/F
Pipe Beta1 nut
Social Social
activity, were used as positive controls for this technique. Additional sections, taken from each specimen block, were stained with hematoxylin and eosin for histopathologic examination. All results were assessedby means of light microscopy. GGT slides were refrigerated to prevent formation of crystallization products, which distort histochemical assessment. The sections were photographed to record histologic findings. For each patient, age, sex, history of tobacco and alcohol use, and clinical diagnosis were recorded separately. RESULTS
Samples were obtained from surgical specimensof sixteen patients, ranging in age from 12 to 83 years. There were twelve casesof known oral squamous cell carcinoma, of which 9 were noted in smokers. One patient was a betel quid chewer, and two were not tobacco users. In addition, a papilloma and a sample of normal mucosa (adjacent to an inflammatory ulcer) were removed from smokers, while a fibroepithelial polyp and mucosa overlying a developmental abnormality were taken from nonsmokers (Table I).
Final diagnosis Fibroepithelial polyp No mucosal lesion Papilloma Inflammatory ulcer Squamous carcinoma, low-grade Squamous carcinoma, alveolar Squamous carcinoma, tonsillar Squamous carcinoma, floor of mouth Squamous carcinoma, floor of mouth Squamous carcinoma, floor of mouth Squamous carcinoma, retromolar tongue Squamous carcinoma, tongue Squamous carcinoma, floor of mouth Squamous carcinoma, floor of mouth Verrucous carcinoma Squamous carcinoma, tongue
Diffuse
+ +
Focal (cellular)
+
+
+
+
+
+
+
+
+
+
+
-
+
+
+
+
+
+
+
+ +
+ +
Diffuse positive staining for GGT activity was evident throughout the epithelium in all samples from the twelve tobacco users (including the betel quid user). This staining was most concentrated in the basal layer (Figs. 1 and 2). Within all twelve carcinomas, individual cells and clumps of cells were heavily stained for GGT activity (Fig. 3). Keratin within the carcinomas also stained postively for GGT activity. Samples taken from the margins of the tumor showed similar heavily GGT-positive cells. In all twelve carcinoma cases multiple samples were taken from tissue beyond the apparent margin of the tumor. Occasional nests of cells were noted to stain positively for GGT activity in the epithelium adjacent to, but not part of, the tumor. The latter were smaller clumps within the basal layers and were less frequent at a distance from the tumor itself. There was little or no evidence of dysplasia or dyskaryosis associated with these histochemical lesions. Occasional heavily GGT-positive cells were encountered in the epithelium distant from the lesion itself in four cases (Figs. 1 and 2); however, in eight cases of carcinoma, no positive cellular staining for GGT activity was noted distant from the region of the
Gamma-glutamyl
Volume64 Number 2
a
transpeptidase activity in carcinoma
1 a”k
199
‘ *
.
-,
Fig. 1. Photomicrograph of mucosa distant from a carcinoma in a smoker, showing both the diffuse GGT positivity (open arrow), most concentrated in the basal layers, and the heavy cellular positivity in individual basal cells and small groups of cells (dark arrows). (Original magnification, X63)
Fig. 2. Higher magnification of the epithelium shown in Fig. 1 demonstrating the markedly GGT-positive individual basal cells (dark arrows), in contrast with the diffusepattern (open arrow).
tumor. When the patient was not a tobacco user, and the sample was not from a malignant lesion, no staining for GGT activity was evident. DISCUSSION
Histochemical examination for GGT activity in these sixteen cases has demonstrated a diffuse increase in activity in the oral epithelium of all tobacco users, differing from a heavy cellular positiv-
ity in the squamous cell carcinomas. These results in carcinomas are comparable to those described by Fiala and coworkers” in carcinomas of the oral cavity, oropharynx, larynx, and esophagus. More recently, Calderon-Solt and SolP examined samples from 46 patients and observed GGT activity in each of the 22 squamouscell carcinomas and 33 dysplastic lesions taken from the oral cavity, pharynx, and larynx. They also noted GGT staining in nonmalig-
200
Mock, Whitestone, and Freeman
Oral S:urg. August ‘987
Fig. 3. Within the carcinomas, individual cells and clumps of cells stained heavily for GGT activity (dark arrows). There is also the diffuse GGT background positivity, commonly seen with smokers (open
arrow).
nant epithelium in eight patients. Two of these apparently had no lesions, two had inflammatory lesions, two had squamous carcinomas elsewhere, one had a dysplastic lesion, and one had a papilloma. All of these patients had a history of tobacco use; however, three of the patients had discontinued the practice at least 6 months prior to surgery. The results of this study are comparable to those of Calderon-Solt & Salt.‘* In our examination, we attempted to distinguish between a diffuse pattern of staining and heavy cellular staining. In all twelve carcinomas, there were focal aggregations of GGTpositive cells. The samples from the one nonuser of tobacco lacked a generalized diffuse pattern of staining and the verrucous carcinoma exhibited GGT positive cells only in the basal half of the epithelium. The staining in the latter case was primarily diffuse with an occasional strong cellular reaction. In this one case it was difficult to differentiate from the diffuse pattern seenin nonmalignant lesions in smokers. Two of the samples of benign lesions showed a diffuse pattern of GGT positivity; one of the lesions was a papilloma. Both of these patients were smokers. Although not clearly defined, tobacco exposure may play a significant role in the expression of GGT. Certainly, as a well-established carcinogenic agent, tobacco may play an initiating and/or promoting role in sequential oral carcinogenesis.21*22 On the other hand, tobacco exposure may produce cellular changes totally unrelated to malignant transformation as well. Therefore, the significance of the diffuse GGT positivity in the oral epithelium of tobacco users is of debatable significance.
The earliest steps in carcinogenesis have been shown not to manifest demonstrable histologic changes. It is likely that subtle biochemical changes are occurring in these early stages. Holley20 has suggested that the early changes include an alteration in the cell surface membrane. The discrete changes in GGT activity as demonstrated by histochemical techniques may reflect such carcinogeninduced alterations in individual cells. The occurrence of mpltiple primary tumors in the patient with oral carcinoma is well recognized, and the tobacco user is at increased risk.23-25 This is quite reasonable, as more than one small area would be exposedto the carcinogenic influence. Therefore, the discrete foci of cells distant from tumors noted in four casesin this study that stained heavily for GGT activity may represent populations of initiated cells from which multiple primary tumors might have ultimately developed. Although speculative at present, it is a reasonable hypothesis. GGT activity may prove to be a useful histochemical marker for preneoplastic cells in oral epithelium. Longitudinal studies are necessary to demonstrate that the histochemical lesions will ultimately develop into squamous carcinomas. More extensive studies are also necessary to demonstrate that the diffuse positive pattern can be reproducibly distinguished from the cellular pattern and that their distinction is significant. REFERENCES
1. Rosalki SB. Gamma-glutamyl transpeptidase. Adv Clin Chem 1975;17:53-107.
Volume 64 Number 2
Gamma-glutamyl
2. Novogrcdsky A, Tate SS, Meister A. Gamma-glutamyl transpeptidase, a lymphoid cell surface marker: relationship to blastogenesis, differentiation and neoplasia. Proc Nat1 Acad Sci USA 1976;73:2414-8. 3. Griffith OW, Bridges RJ, Meister A. Transport of gammaglutamyl amino acids: role of glutathione and gammaelutamvl transeeotidase. Proc Nat1 Acad Sci USA 1979; ?6:6319-22. . 4. Solt DB, Medline A, Farber E. Rapid emergence of carcinogen-induced hyperplastic lesions in a new model for the sequential analysis of liver carcinogenesis. Am J Path01 1977;88:595-618. 5. Pugh TD, Goldfarb S. Quantitative histochemical and autoradiographic studies of hepatocarcinogenesis in rats fed 2acetylaminofluorine followed by phenobarbital. Cancer Res 1978;38:4450-7. 6. Ogawa K, Solt DB, Farber E. Phenotypic diversity as an early property of putative preneoplastic hepatocyte populations in liver carcinogen&s. Cancer Res 1980;40:725-30. I. Fiala S, Fiala AE, Dixon B. Gamma-glutamyl transpeptidase in transplantable, chemically induced rat hepatomas and “spontaneous” mouse hepatomas. J Nat1 Cancer Inst 1972;48:1393-401. 8. De Young LM, Richards WL, Bonzelet W, Tsai LL, Boutwell RK. Localization and significance of gamma-glutamyltranspeptidase in normal and neoplastic mouse skin. Cancer Res 1978;38:3697-701. 9. Albert Z. Gamma-glutamyl transpeptidase in cancers of different human organs. Nature 1965;205:407. 10. Fiala S, Trout E. Pragani B, Fiala ES. Increased gammaglutamyl transferase activity in human colon cancer. Lancet 1979;1:1145. -11 Bernard P, McPartland K, Hassan M, Solt D. Investigation of the carcinogen-specific nature of gamma-glutamyl transpeptidase (GGT)-rich cells chemically induced in hamster buccal pouch epithelium (HBPE). AACR Proceedings, 1982, Abstr. 211. 12. Solt D, Solt L, Odajima T. Induction of gamma-glutamyl transpeptidase (GGT)-rich foci and carcinomas in N-methylN-benzylnitrosamine treated hamster buccal pouch epitheliurn. AACR Proceedings, 1983, Abstr. 209. 13. Bernard DP, Solt DB. Carcinogen specificity of gammaglutamyl transpeptidase-rich foci chemically induced in hamster buccal pouch epithelium. In: Safety Evaluation and Regulations of Chemicals, First International Conference, Basel: S. Karger, 1982:173-82.
transpeptidase activity in carcinoma
201
14. Solt DB. Localization of gamma-glutamyl transpeptidase in hamster buccal pouch epithelium treated with 7, 12 dimethylbenzanthracene. J Nat1 Cancer Inst 1981;67:193-200. 15. Solt DB, Shklar G. Rapid induction of gamma-glutamyl transpeptidase rich intra-epithelial clones in 7, 12 dimethylbez(a)anthracene treated hamster buccal pouch. Cancer Res 1982;42:285-91. 16. Rutenburg AM, Kim H, Fischbein JW, Hanker JS, Wasserkrug HL, Seligman AM. Histochemical and ultrastructural demonstration of y-glutamyl transpeptidase activity. J Histothem Cytochem 1969;17:517-26. 17. Fiala S, Trout EC, Teague CA, Fiala ES. Gamma-glutamyltransferase: a common marker of human epithelial tumors? Cancer Detect Prev 1980;3:471-85. 18. Calderon-Solt L, Solt DB. Gamma-glutamyl transpeptidase in precancerous lesions and carcinomas of oral, pharyngeal and laryngeal mucosa. Cancer 1985;56:138-43. 19. Farber E, Cameron R. The sequential analysis of cancer development. Adv Cancer Res 1980;31:125-226. 20. Holley RW. A unifying hypothesis concerning the nature of malignant growth. Proc Nat1 Acad Sci USA 1972;69:28401. 21. Wynder EL, Stellman SD. Comparative epidemiology of tobacco-related cancers. Cancer Res 1977;37:4608-22. 22. Smith EM. Epidemiology of oral and pharyngeal cancers in the United States: review of recent literature. J Nat1 Cancer Inst 1979;63:1189-98. 23. Silverman S, Griffith M. Smoking characteristics of patients with oral carcinoma and the risk for second oral primary carcinoma. J Am Dent Assoc 1972;85:637-40. 24. Moertel GG. Multiple primary malignant neoplasms:historical perspectives. Cancer 1977;40:1786-92. 25. Sharp GS, Bulock WK, Helsper JT. Multiple oral carcinomas. Cancer 1961;14:512-6. Reprint requests to
Dr. D. Mock Faculty of Dentistry University of Toronto 124 Edward St. Toronto, Ontario M5G lG6, Canada
SINAI
and J. Freeman,***
Toronto, Ontario, Canada
HOSPITAL
Gamma-glutamyl transpeptidase (GGT) activity has been shown to increase in preneoplastic cells in various experimental carcinogenesis models and in some human tumors. This pilot study was undertaken to examine its potential use in detection of sites of human oral carcinogenesis. Samples were taken from 16 surgical specimens: 12 resection specimens for squamous carcinomas and 4 benign mucosal lesions. Twelve of the patients were tobacco users. Diffuse GGT activity was found in all tobacco users and heavy cellular activity in all carcinomas. In four of the carcinoma-resection specimens, occasional cells staining heavily for GGT activity were noted in epithelium distant from the tumor itself. These results are discussed in relation to the potential value of GGT activity as a marker from preneoplastic changes in oral epithelium. (ORAL SURG. ORAL MED. ORAL PATHOL. 1987;64:197-201)
Gamma-glutamyl transpeptidase (GGT) is a membrane-bound enzyme found in epithelial cells of jejunal villi, pancreatic acini, bile ducts, seminal vesicles, the choroid plexus, and proximal convoluted tubules of kidney,’ as well as in lymphocytes.* This glycoprotein catalyzes the transfer of gamma-glutamy1 moiety of peptides to other peptides and amino acids. Therefore, the physiologic role of GGT is thought to be related to translocation of amino acids across cell membranes.3 GGT has proved useful as a histochemical marker for preneoplastic cells in the rat liver:5 in experimentally induced hepatomas,6*7and in neoplastic mouse skin.* Limited studies have reported increased GGT activity in some human cancers.9*LoIn untreated hamster cheek pouch mucosa and in hamster pouch mucosa treated with noncarcinogenic irritants, GGT activity was not evident,11-‘3In epithelial whole mounts of DMBA-exposed hamster cheek pouch mucosa, GGT was detected histochemically in localized areas of dysplasia and in well-differentiated squamous cell carcinomas.‘4vIs These preliminary studies suggest that GGT may be useful as a marker in oral squamous cell carcinomas and precancerous lesions.
*Department of Pathology and Dentistry. **Department of Dentistry. ***Department of Otolaryngology.
MATERIALS AND METHODS Tissue samples
Mucosal samples were obtained from 16 patients undergoing diagnostic or therapeutic surgical procedures of the oral cavity. Included were only those patients for whom adequate documentation of smoking, alcohol intake, previous related biopsies, and relevant clinical history was available. Multiple samples were taken, including both neoplastic tissue and adjacent grossly unremarkable tissue, from the tissue resection specimens. Tissue samples were obtained immediately after resection of the relevant area. The tissueswere quick-frozen, and cryostat-cut sections 5 pm in thickness were obtained. Histochemistry
The cryostat-cut sections of fresh frozen tissue were mounted on glass slides. Histochemical localization of GGT activity was carried out according to the method of Rutenburg and coworkers.L6 The sections were incubated for 30 minutes at room temperature in fresh medium containing gammaglutamyl-Cmethoxy 2 naphthylamide, fast blue BB salt, and glycylglycine. After rinsing with 0.85% sodium chloride, the tissues were dipped in 0.2 mol/L cupric sulfate solution, rinsed again in 0.85% sodium chloride, and washed in distilled water. The sections were counterstained with hematoxylin. Slides were coverslipped with glycerin gelatin mounting medium. Sections of kidney, rich in GGT 197
198
Mock, Whitestone, and Freeman
Oral Surg. August 1987
Table I GGT staining Patient No.
Age/Sex
Tobacco use
Alcohol
use
25/M 12/F 26/M 45/F 54/F
None None Cigarette Cigarette None
Social None Social Social Moderate
70/F
Cigarette
Moderate
7
50/F
Cigarette
8
77/M
Cigarette
Heavy
9
66/M
Cigarette
Moderate
10
56/M
Cigarette
Heavy
11
83/F
None
Social
12
65/F
Cigarette
Heavy
13
39/F
Cigarette
Moderate
14
37/M
Cigarette
Heavy
15 16
58/M 59/F
Pipe Beta1 nut
Social Social
activity, were used as positive controls for this technique. Additional sections, taken from each specimen block, were stained with hematoxylin and eosin for histopathologic examination. All results were assessedby means of light microscopy. GGT slides were refrigerated to prevent formation of crystallization products, which distort histochemical assessment. The sections were photographed to record histologic findings. For each patient, age, sex, history of tobacco and alcohol use, and clinical diagnosis were recorded separately. RESULTS
Samples were obtained from surgical specimensof sixteen patients, ranging in age from 12 to 83 years. There were twelve casesof known oral squamous cell carcinoma, of which 9 were noted in smokers. One patient was a betel quid chewer, and two were not tobacco users. In addition, a papilloma and a sample of normal mucosa (adjacent to an inflammatory ulcer) were removed from smokers, while a fibroepithelial polyp and mucosa overlying a developmental abnormality were taken from nonsmokers (Table I).
Final diagnosis Fibroepithelial polyp No mucosal lesion Papilloma Inflammatory ulcer Squamous carcinoma, low-grade Squamous carcinoma, alveolar Squamous carcinoma, tonsillar Squamous carcinoma, floor of mouth Squamous carcinoma, floor of mouth Squamous carcinoma, floor of mouth Squamous carcinoma, retromolar tongue Squamous carcinoma, tongue Squamous carcinoma, floor of mouth Squamous carcinoma, floor of mouth Verrucous carcinoma Squamous carcinoma, tongue
Diffuse
+ +
Focal (cellular)
+
+
+
+
+
+
+
+
+
+
+
-
+
+
+
+
+
+
+
+ +
+ +
Diffuse positive staining for GGT activity was evident throughout the epithelium in all samples from the twelve tobacco users (including the betel quid user). This staining was most concentrated in the basal layer (Figs. 1 and 2). Within all twelve carcinomas, individual cells and clumps of cells were heavily stained for GGT activity (Fig. 3). Keratin within the carcinomas also stained postively for GGT activity. Samples taken from the margins of the tumor showed similar heavily GGT-positive cells. In all twelve carcinoma cases multiple samples were taken from tissue beyond the apparent margin of the tumor. Occasional nests of cells were noted to stain positively for GGT activity in the epithelium adjacent to, but not part of, the tumor. The latter were smaller clumps within the basal layers and were less frequent at a distance from the tumor itself. There was little or no evidence of dysplasia or dyskaryosis associated with these histochemical lesions. Occasional heavily GGT-positive cells were encountered in the epithelium distant from the lesion itself in four cases (Figs. 1 and 2); however, in eight cases of carcinoma, no positive cellular staining for GGT activity was noted distant from the region of the
Gamma-glutamyl
Volume64 Number 2
a
transpeptidase activity in carcinoma
1 a”k
199
‘ *
.
-,
Fig. 1. Photomicrograph of mucosa distant from a carcinoma in a smoker, showing both the diffuse GGT positivity (open arrow), most concentrated in the basal layers, and the heavy cellular positivity in individual basal cells and small groups of cells (dark arrows). (Original magnification, X63)
Fig. 2. Higher magnification of the epithelium shown in Fig. 1 demonstrating the markedly GGT-positive individual basal cells (dark arrows), in contrast with the diffusepattern (open arrow).
tumor. When the patient was not a tobacco user, and the sample was not from a malignant lesion, no staining for GGT activity was evident. DISCUSSION
Histochemical examination for GGT activity in these sixteen cases has demonstrated a diffuse increase in activity in the oral epithelium of all tobacco users, differing from a heavy cellular positiv-
ity in the squamous cell carcinomas. These results in carcinomas are comparable to those described by Fiala and coworkers” in carcinomas of the oral cavity, oropharynx, larynx, and esophagus. More recently, Calderon-Solt and SolP examined samples from 46 patients and observed GGT activity in each of the 22 squamouscell carcinomas and 33 dysplastic lesions taken from the oral cavity, pharynx, and larynx. They also noted GGT staining in nonmalig-
200
Mock, Whitestone, and Freeman
Oral S:urg. August ‘987
Fig. 3. Within the carcinomas, individual cells and clumps of cells stained heavily for GGT activity (dark arrows). There is also the diffuse GGT background positivity, commonly seen with smokers (open
arrow).
nant epithelium in eight patients. Two of these apparently had no lesions, two had inflammatory lesions, two had squamous carcinomas elsewhere, one had a dysplastic lesion, and one had a papilloma. All of these patients had a history of tobacco use; however, three of the patients had discontinued the practice at least 6 months prior to surgery. The results of this study are comparable to those of Calderon-Solt & Salt.‘* In our examination, we attempted to distinguish between a diffuse pattern of staining and heavy cellular staining. In all twelve carcinomas, there were focal aggregations of GGTpositive cells. The samples from the one nonuser of tobacco lacked a generalized diffuse pattern of staining and the verrucous carcinoma exhibited GGT positive cells only in the basal half of the epithelium. The staining in the latter case was primarily diffuse with an occasional strong cellular reaction. In this one case it was difficult to differentiate from the diffuse pattern seenin nonmalignant lesions in smokers. Two of the samples of benign lesions showed a diffuse pattern of GGT positivity; one of the lesions was a papilloma. Both of these patients were smokers. Although not clearly defined, tobacco exposure may play a significant role in the expression of GGT. Certainly, as a well-established carcinogenic agent, tobacco may play an initiating and/or promoting role in sequential oral carcinogenesis.21*22 On the other hand, tobacco exposure may produce cellular changes totally unrelated to malignant transformation as well. Therefore, the significance of the diffuse GGT positivity in the oral epithelium of tobacco users is of debatable significance.
The earliest steps in carcinogenesis have been shown not to manifest demonstrable histologic changes. It is likely that subtle biochemical changes are occurring in these early stages. Holley20 has suggested that the early changes include an alteration in the cell surface membrane. The discrete changes in GGT activity as demonstrated by histochemical techniques may reflect such carcinogeninduced alterations in individual cells. The occurrence of mpltiple primary tumors in the patient with oral carcinoma is well recognized, and the tobacco user is at increased risk.23-25 This is quite reasonable, as more than one small area would be exposedto the carcinogenic influence. Therefore, the discrete foci of cells distant from tumors noted in four casesin this study that stained heavily for GGT activity may represent populations of initiated cells from which multiple primary tumors might have ultimately developed. Although speculative at present, it is a reasonable hypothesis. GGT activity may prove to be a useful histochemical marker for preneoplastic cells in oral epithelium. Longitudinal studies are necessary to demonstrate that the histochemical lesions will ultimately develop into squamous carcinomas. More extensive studies are also necessary to demonstrate that the diffuse positive pattern can be reproducibly distinguished from the cellular pattern and that their distinction is significant. REFERENCES
1. Rosalki SB. Gamma-glutamyl transpeptidase. Adv Clin Chem 1975;17:53-107.
Volume 64 Number 2
Gamma-glutamyl
2. Novogrcdsky A, Tate SS, Meister A. Gamma-glutamyl transpeptidase, a lymphoid cell surface marker: relationship to blastogenesis, differentiation and neoplasia. Proc Nat1 Acad Sci USA 1976;73:2414-8. 3. Griffith OW, Bridges RJ, Meister A. Transport of gammaglutamyl amino acids: role of glutathione and gammaelutamvl transeeotidase. Proc Nat1 Acad Sci USA 1979; ?6:6319-22. . 4. Solt DB, Medline A, Farber E. Rapid emergence of carcinogen-induced hyperplastic lesions in a new model for the sequential analysis of liver carcinogenesis. Am J Path01 1977;88:595-618. 5. Pugh TD, Goldfarb S. Quantitative histochemical and autoradiographic studies of hepatocarcinogenesis in rats fed 2acetylaminofluorine followed by phenobarbital. Cancer Res 1978;38:4450-7. 6. Ogawa K, Solt DB, Farber E. Phenotypic diversity as an early property of putative preneoplastic hepatocyte populations in liver carcinogen&s. Cancer Res 1980;40:725-30. I. Fiala S, Fiala AE, Dixon B. Gamma-glutamyl transpeptidase in transplantable, chemically induced rat hepatomas and “spontaneous” mouse hepatomas. J Nat1 Cancer Inst 1972;48:1393-401. 8. De Young LM, Richards WL, Bonzelet W, Tsai LL, Boutwell RK. Localization and significance of gamma-glutamyltranspeptidase in normal and neoplastic mouse skin. Cancer Res 1978;38:3697-701. 9. Albert Z. Gamma-glutamyl transpeptidase in cancers of different human organs. Nature 1965;205:407. 10. Fiala S, Trout E. Pragani B, Fiala ES. Increased gammaglutamyl transferase activity in human colon cancer. Lancet 1979;1:1145. -11 Bernard P, McPartland K, Hassan M, Solt D. Investigation of the carcinogen-specific nature of gamma-glutamyl transpeptidase (GGT)-rich cells chemically induced in hamster buccal pouch epithelium (HBPE). AACR Proceedings, 1982, Abstr. 211. 12. Solt D, Solt L, Odajima T. Induction of gamma-glutamyl transpeptidase (GGT)-rich foci and carcinomas in N-methylN-benzylnitrosamine treated hamster buccal pouch epitheliurn. AACR Proceedings, 1983, Abstr. 209. 13. Bernard DP, Solt DB. Carcinogen specificity of gammaglutamyl transpeptidase-rich foci chemically induced in hamster buccal pouch epithelium. In: Safety Evaluation and Regulations of Chemicals, First International Conference, Basel: S. Karger, 1982:173-82.
transpeptidase activity in carcinoma
201
14. Solt DB. Localization of gamma-glutamyl transpeptidase in hamster buccal pouch epithelium treated with 7, 12 dimethylbenzanthracene. J Nat1 Cancer Inst 1981;67:193-200. 15. Solt DB, Shklar G. Rapid induction of gamma-glutamyl transpeptidase rich intra-epithelial clones in 7, 12 dimethylbez(a)anthracene treated hamster buccal pouch. Cancer Res 1982;42:285-91. 16. Rutenburg AM, Kim H, Fischbein JW, Hanker JS, Wasserkrug HL, Seligman AM. Histochemical and ultrastructural demonstration of y-glutamyl transpeptidase activity. J Histothem Cytochem 1969;17:517-26. 17. Fiala S, Trout EC, Teague CA, Fiala ES. Gamma-glutamyltransferase: a common marker of human epithelial tumors? Cancer Detect Prev 1980;3:471-85. 18. Calderon-Solt L, Solt DB. Gamma-glutamyl transpeptidase in precancerous lesions and carcinomas of oral, pharyngeal and laryngeal mucosa. Cancer 1985;56:138-43. 19. Farber E, Cameron R. The sequential analysis of cancer development. Adv Cancer Res 1980;31:125-226. 20. Holley RW. A unifying hypothesis concerning the nature of malignant growth. Proc Nat1 Acad Sci USA 1972;69:28401. 21. Wynder EL, Stellman SD. Comparative epidemiology of tobacco-related cancers. Cancer Res 1977;37:4608-22. 22. Smith EM. Epidemiology of oral and pharyngeal cancers in the United States: review of recent literature. J Nat1 Cancer Inst 1979;63:1189-98. 23. Silverman S, Griffith M. Smoking characteristics of patients with oral carcinoma and the risk for second oral primary carcinoma. J Am Dent Assoc 1972;85:637-40. 24. Moertel GG. Multiple primary malignant neoplasms:historical perspectives. Cancer 1977;40:1786-92. 25. Sharp GS, Bulock WK, Helsper JT. Multiple oral carcinomas. Cancer 1961;14:512-6. Reprint requests to
Dr. D. Mock Faculty of Dentistry University of Toronto 124 Edward St. Toronto, Ontario M5G lG6, Canada