215
Cancer Letters, 65 (1992) 215 - 220 Elsevier Scientific Publishers Ireland Ltd.
Oral cancer progression expression
and c-erbB-Z/neu
Lin Houa, Daren Shib, Shi-Ming TuC, Hua-Zhong Disheng Linga ‘Department Shanghai
of Oral Pathology,
Medical
Uniuersity
University of Texas, M.D.
Shangdong
Medical
Cancer Hospital,
Anderson
Cancer
Uniuersity,
Shanghai Center,
200032
Houston,
proto-oncogene
Zhang”,
Mien-Chie
Jinan 250012, (P.R.
China)
Texas 77030
bDepartment
HungC and
of Immunopathology,
and ‘Department
of Tumor
Biology,
(USA)
(Received 30 April 1992) (Revision received 8 June 1992) (Accepted
9 June 1992)
Summary
Introduction
Monoclonal
antibody in staining of 86 human mucosa. These tissue a spectrum from 7
protein
was utilized
hyperplasia,
15 mild
PAb3 to c-erbB-2/neu immunoperoxidase specimens from oral specimens represented the
normal dysplasia,
dysplasia, 20 severe squamous cell carcinoma. that as the cells acquire
to
9
simple
14
moderate dysplasia and 21 Our study indicated
a more malignant phenotype, there was a progressive increase in neu expression. It also suggested that neu may be involved in the development of oral cancers and that its evaluation in the early stages may assist in the diagnosis and management of oral
cancers.
oncogenes; Keywords: carcinoma; (HER-Z) ; oral chemistry
c-erbB-Z/neu immunohisto-
Correspondence to: Mien-Chie Hung, Dept. of Tumor Biology, M.D. Anderson Cancer Center, Box 79, 1515 Holcombe Blvd., Houston, TX 77030, USA.
0304-3835/92/$05.00 Printed and Published
0 1992 Elsevier Scientific Publishers in Ireland
C-erbB-Z/neu gene (also known as HER-2) was originally identified in neuroblastomas which developed in the offsprings of ethylnitrosourea injected pregnant rats [ 131. Subsequently, it was found to be homologous to the v-erbB oncogene and was cloned using v-erbB as a probe [7]. Neu is a 185kDa transmembrane protein with tyrosine kinase activity. It is highly homologous to but distinct from the epidermal growth factor receptor, EGFR. Activation of neu can occur by a single mutation in the transmembrane domain [2]. However, the pathogenesis of human malignancies appear to involve the overexpression of the normal neu gene product. In cases where there is neu without significant gene overexpression amplification, transcriptional and/or translational dysregulation may be responsible for neu overexpression [9]. Neu amplification has been detected in a variety of human primary tumors, including salivary adenocarcinoma [14], gastric carcinoma [4], colon carcinoma [3], lung carcinoma [12], ovarian carcinoma [17,24] and breast carcinoma [8,16]. These observations suggest that neu may be playing Ireland Ltd
216
an important role in the process of tumorigenesis. Furthermore, neu may also be involved in the processes of metastases. For instance, neu overexpression has been shown to correlate with lymph node metastases in breast and lung cancers [lo, 15,16,20]. Oral cancers as defined by the WHO (World Health Organization) is one of the 10 most frequent cancers in the world. The majority of these cases arose in the developing countries. Although oral cancer is relatively less common in the westernized countries, geographic heterogeneity is evident in certain areas. The role of cytokines in the growth and progression of squamous cell cancers has been studied recently. TGF-cr has been shown to have sequence homology to EGF and binds to EGFR. It appears to be involved in the growth and progression of these tumors [23]. TGF-fl and interleukin-1 have also been found to play important roles in the development of squamous cell cancers [ll], In addition, there is evidence for cross interaction between neu and EGFR [19] suggesting a role for neu in these tumors. This study was designed to investigate the role of neu in the development and progression of oral cancers. We analyzed the expression and distribution of neu protein in various tissues ranging from normal to progressively more malignant phenotypes. The evidence suggests that neu expression is associated with malignancy of the oral cavity. Materials
and Methods
Specimens All specimens were obtained from the Department of Oral Pathology of Shandong Medical University. These specimens were routinely fixed in formalin and embedded in paraffin, then serial sections with thickness of 5 pm were made. The diagnoses of epithelial dysplasia were made according to the 12 criteria put forth by WHO [22] and the criteria of Banoczy [l]. Eighty-six specimens were examined, including 9 cases of simple hyperplasia (6 leukoplakia, 3 lichen planus), 15 cases of mild dysplasia (12 leucoplakia, 2
lichen planus, 1 rhomboid glossitis), 14 cases of moderate dysplasia (11 leukoplakia, 1 lichen planus, 1 erythroplasia, 1 abnormal proliferation of submucosal fibers), 21 cases of invasive squamous cell carcinoma and 7 cases of normal oral mucosa. Reagent Monoclonal antibody c-neuMAb3 of c-erbB2 product was supplied by Oncogene Science Inc. Manhasset, NY, Biotinylated horse antimouse IgG and ABC kit were supplied by Vector Lab., Burlingame, CA. Method Immunoperoxidase staining technique was derived from Hsu’s ABC technique [5,6]. The procedure is as follows: (1) slides undergo deparaffination and hydration in gradient of alcohol; (2) blocked in 3% HsOs for 15 min, washed in TBS (0.05 M, pH 7.6); (3) digested in 0.05% trypsin for 20 min, washed in TBS; (4) treated in 10% horse serum for 20 min; (5) incubated with PAb3 (1: 100 or 10 pg/ml) for 60 min, then kept at 4OC overnight, washed in TBS; (6) incubated with biotinylated horse anti-mouse IgG (1:200) for 30 min, washed in TBS; (7) incubated with ABC reagent (1: 100) for 60 min, washed in TBS; (8) developed in 0.04% DAB-HsOs (40 mg of DAB in 100 ml of TBS, add 20 ~1 of 3% H202), washed in tap water; (9) stained with hematoxylin, dehydrated in alcohol, cleaned in xylol and mounted with resin. All procedures were performed at room temperature unless otherwise indicated. Negative controls in which TBS replaced the primary antibody were run with each batch of stains. A previously identified strongly staining tumor was used as a positive control. Intra and inter assay consistency was maintained by running these positive and negative controls with each batch of staining. Criteria of results The prepared slides were observed under optic microscope. It was assigned as positive for cells with brown granules and as negative for cells without brown granules.
217
.. t
, t. I*
1 . @,
A
oral cavity, (B) and (C) human oral Fig. 1. Immunohistochemical staining of ~185 neu in (A) normal human squamous cell carcinoma. Representative sections were stained with c-neu MAb3 monoclonal antibody at magnifications of x 100 (A and B) and x 400 (C).
218
Table I.
The relationship
Histol grades
between
histologic grades and the expression
Expression
of c-neu mAb3
of c-neu mAb3 in basal cell layer.
Rate of positive (%I
Normal oral mucosa Simple hyperplasia Mild dysplasia Moderate dysplasia Severe dysplasia Invasive carcinoma
n
+
-
7 9 15 14 20 21
0 0 2 10 16 21
7 9 13 4 4 0
Statistical method The data were analyzed using the test for a linear trend in proportions [18] and x2 test.
There was no significant difference between the basal cell layer and the prickle cell layer in the expression of neu. The increase in the positive rate of c-neuMAb3 expression, the intensity of c-neuMAb3 staining and the number of positive cells was noted to correlate with the progression of the oral tissue from dysplasia to carcinoma.
Results We did not detect expression of neu product in normal oral mucosa (Fig. la) and in simple hyperplasia (except in the prickle cell layer of a case of simple hyperplasia) . Neu product expression gradually becomes more evident and increases from mild to moderate and to severe dysplasia (linear trend test, P < 0.001). The increment is most remarkable and statistically significant in the transition from mild to moderate dysplasia (P < 0.05). Positive expression was also noted in all squamous cell carcinoma examined (Figs. lb, lc). The relationship between histologic grades of the oral specimens and the expression of neu as detected by c-neuMAb3 in the basal cell layer and in prickle cell layer was shown in Table I and Table II. Table II.
The relationship
Histol grades
between
0 0 13 71 80 100
Discussion Recent studies have indicated that malignant transformation may involve a multi-step process and that many if not all of these steps may implicate structural anomalies or regulatory dysfunction of oncogenes (or tumor suppressor genes). Oral cancers are more common in men than women. Smoking and alcoholic consumption are known risk factors for oral cancers. Moreover, certain papillomaviruses may also contribute to the development of oral cancers. Though the molecular mechanisms of action of these environmental
histologic grades and the expression
Expression
of c-neu mAb3
Rate of positive (%I
Normal oral mucosa Simple hyperplasia Mild dysplasia Moderate dysplasia Severe dysplasia Invasive carcinoma
n
+
7 9 15 14 20 21
0 1 6 11 17 21
of c-neu mAb3 in prickle cell layer.
7 8 9 3 3 0
0 11 40 79 85 100
219
risk factors are still largely unknown, recent studies have suggested that TGF-cr and IL-1 may be some of the involved mediators. Walker [Zl] made the original observation in 1989 that immunohistochemical staining of human breast cancer with polyclonal antiserum 21N against neu protein was able to distinguish tumor cells from normal breast epithelium. It was also demonstrated that staining was associated with positive nodal status and poorer nuclear grade thereby suggested the importance of immunohistochemistry in the diagnosis and prognosis of these cancers. This paper provides evidences for a potential relationship between neu protein overexpression and the malignant transformation of oral cancers. (1) The staining of simple hyperplasia and mild dysplasia of epithelium was similar to that of normal oral mucosa. It implied that there was no neu overexpression in these lesions. (2) The intensity of staining and the number of positive cells increased markedly from mild dysplasia to moderate and severe dysplasia. The phenomenon was even more pronounced in carcinomas. This implied neu overexpression in these later lesions. The degree of staining intensity and the number of positive cells discriminate moderate and severe dysplasia of the oral epithelium from normal and simple dysplastic mucosa. It may provide reliable and accurate criteria to diagnose precancerous lesions of the oral mucosa. (3) It was observed that cancer cells from all 21 cases of squamous cell carcinoma showed strong staining implying neu overexpression. Whether neu overexpression is the consequence of gene amplification or transcriptional/translational dysregulation needs further experimentation. The variation of staining by c-neuMAb3 in the carcinoma ceils suggests possible tumor heterogeneity. (4) A tendency towards increased neu product in progressively more advanced cancer was evident from our results. Assuming the degree of staining increased proportionally to the amount of neu product, the presence of neu may be involved in cell dedifferentiation.
(5) Finally, th e staining pattern of severe dysplasia of oral epithelium resembled that of carcinoma. The strong morphological similarity and the identical pattern of neu expression support the argument that moderate/severe dysplasia have a high risk of developing malignancy in precancerous lesions. As far as we know, this study provides the first evidence for a significant association between neu oncogene overexpression and oral cancer progression. By means of an immunohistochemical approach using monoclonal antibodies against neu protein, we demonstrated that there is overexpression of neu in more advanced stages of oral cancer. Whether overexpression of neu is due to gene amplification or due to transcriptional/translational dysregulation of a normal neu product is not known. That neu may be a discrete anomaly contributing to the multistep process of malignant transformation in oral cancer is a distinct possibility. This opens a novel avenue to the basic study and early diagnosis of oral cancer.
Acknowledgement
This work was partially supported Smokeless Tobacco Research Council No. 0287 to M.C. Hung. We would thank Terry Smith for her assistance statistical analysis.
by the Grant like to in our
References Banouy,
J. and Csiba, A. (1976)
Occurrence
dysplasia in oral leukoplakia.
Oral Surg.,
Bargmann,
and Weinberg,
C.I.,
Hung, M.C.
Multiple independent mutation
~185.
Cell, 45, 649-657.
D’Emilia,
altering
the
transmembrane
J., Bulovas, K., D’Ercole,
and Summerhayes,
erbB-2
gene
neoplastic
R.A.
(1986)
activations of the neu oncogene by a
point
J.G.
of epithelial
42, 766 - 774.
product
progression
I.C.
(1989)
of
K., Wolf, B., Steele, Expression of the c-
(~185)
at
in
colon.
the
domain
different
stages
Oncogene,
of
4,
1233-1239. Fukushige, S.-l., Matsubara, K.-l., Yoshida, M., Sasaki, M., Suzuki, T., Semba, K., Toyoshima. K. and Yamamoto, T. (1986) Localization of a novel v-erbBrelated
gene,
c-erbB-2,
on
chromosome
17
and
its
amplification in a gastric cancer cell line. Mol. Cell Biol., 6,
955-958.
220 5
6
7
8
9
10
11 12
13
14
15
Hsu, S.M. and Raine, L. (1982) Discovery of a high affinlty of phaseolus vulgarts agglutinin (PHA) with gastrosecreting cells. Am. J. Clin. Pathol., 77, 396-400. Hsu, S.M., Raine, L. and Fanger, H. (1981) Use of avidinbiotin-peroxidase complex (ABC) in immunoperoxidase technique: a comparison between ABC and unlabeled antibody (PAP) procedures. J. Histochem. Cytochem., 29, 577-80. Hung, M.C., Schechter, A.L., Chevray, P., Stern, D.F. and Weinberg, R.A. (1986) Molecular cloning of the neu gene: absence of gross structural alteration in oncogenic alleles. Proc. Natl. Acad. Sci. U.S.A., 83, 261- 264. King, CR., Kraus, M.H. and Aaronson, S.A. (1985) Amplification of a novel v-erbB-related gene in a human mammary carcinoma. Science, 229, 974 - 976. Kraus, M.H., Popescu, N.C., Amsbaugh, S.C. and King, C.R. (1987) Overexpression of the EGF receptor-related proto-oncogene erbB-2 in human mammary tumor cell lines by different molecular mechanisms. EMBO J., 6, 605 - 610. Lacroix, H., lglehart, J.D., Skinner, M.A. and Kraus, M.H. (1989) Overexpression of erbB-2 or EGF receptor proteins present in early stage mammary carcinoma is detected simultaneously in matched primary tumors and regional metastases. Oncogene, 4, 145 - 151. Schantz, S.P (1990) Experimental Head and Neck Oncology. Curr. Opin. Oncol., 2, 546-551. Schneider, P.M., Hung, M.C., Chiocca, S.M., Manning, J., Zhao, X.Y., Fang, K. and Roth, J.A. (1989) Differential expression of c-erbB-2 gene in human small cell lung cancer. Cancer Res., 49, 4968-4971. Schubert, D., Heinemann, S., Carlisle, W., Tarikas, H., Kimes, B., Patrick, J., Steinbach, J.H., Gulp, W. and Brandt, B.L. (1974) Clooal cell lines from the rat central nervous system. Nature, 249, 224 - 227. Semba, K., Kamata, N., Toyoshima, K. and Yamamoto, T. (1985) A v-erbB-related protooncogene, c-erbB-2, is distinct from the c-erbB-l/epidermal growth factor receptor gene and is amplified in human salivary gland adenocarcinoma. Proc. Natl. Acad. Sci. U.S.A., 82, 6497-6501. Shi, D., He, G., Cao, S., Pan, W., Zhang, H., Yu, D. and
16
17
18
19
20
21
22
23
24
Hung, M.-C. (1992) Overexpression of the c-erbB-Z/neuencoded ~185 protein in primary lung cancer. Mol. Carcinogen., in press. Slamon, D.J., Clark, G.M., Wong, S.G., Levin, W.J., Ullrich, A. and McGuire, W.L. (1987) Human breast cancer: correlation of relapse and survival with amplification of the HER-Z/neu oncogene. Science, 240, 177- 182. Slamon, D.J., Godolphin, W., Jones, L.A., Holt, J.A., Wong, S.G., Keith, D.E., Levin, W.J., Stuart, S.G., Udove, J., Ullrich, A. and Press, M.F. (1989) Studies of the HER-Z/neu proto-oncogene in human breast and ovarian cancer. Science, 244, 707 - 771. Snedecor, G.W. and Cochran, W.G. (1980) In: Statistical Methods, Seventh Edition, pp. 206 - 208. The Iowa State University Press, Ames, Iowa. Stern, D.F., Kamps, M.P. and Cao, H. (1988) Oncogenic activation of pl85neu stimulates tyrosine phosphorylation in vivo. Mol. Cell Biol., 8, 3969-3973. Tauchi, K., Hori, S., Itoh, H., Osamura, R.Y., Takuda, Y. and Tajima, T. (1989) Immunohistochemical studies on oncogene products (c-erbB-2, EGFR, c-myc) and estrogen receptor in benign and malignant breast lesions, with special reference to their prognostic significance in carcinoma. Virchows Arch., 416, 65 - 73. Walker, R.A., Senior, P.V., Jones, J.L., Critchley, D.R. and Varley, J.M. (1989) An immunohistochemical and insitu hybridization study of c-myc and c-erbB-2 expression in primary human breast carcinoma. J. Pathol., 158, 97 - 105. WHO (1978) WHO Collaborating Center for oral precancerous lesions: definition of leukoplakia and related lesions: an aid to studies on oral precancer. Oral Surg., 46, 518 - 539. Wong, D.T.W., Gallagher, G.T., Gertz, R., Chang, A.L.C. and Shklar, G. (1988) Transforming growth factor alpha in chemically transformed oral keratinocytes. Cancer Res., 48, 3130-3134. Zhang, X., Silvia. E., Gershenson, D. and Hung, M.C. (1989) Amplification and rearrangement of c-erb B protooncogenes in cancer of human female genital tract. Oncogene, 4, 985 - 989.
Cancer Letters, 65 (1992) 215 - 220 Elsevier Scientific Publishers Ireland Ltd.
Oral cancer progression expression
and c-erbB-Z/neu
Lin Houa, Daren Shib, Shi-Ming TuC, Hua-Zhong Disheng Linga ‘Department Shanghai
of Oral Pathology,
Medical
Uniuersity
University of Texas, M.D.
Shangdong
Medical
Cancer Hospital,
Anderson
Cancer
Uniuersity,
Shanghai Center,
200032
Houston,
proto-oncogene
Zhang”,
Mien-Chie
Jinan 250012, (P.R.
China)
Texas 77030
bDepartment
HungC and
of Immunopathology,
and ‘Department
of Tumor
Biology,
(USA)
(Received 30 April 1992) (Revision received 8 June 1992) (Accepted
9 June 1992)
Summary
Introduction
Monoclonal
antibody in staining of 86 human mucosa. These tissue a spectrum from 7
protein
was utilized
hyperplasia,
15 mild
PAb3 to c-erbB-2/neu immunoperoxidase specimens from oral specimens represented the
normal dysplasia,
dysplasia, 20 severe squamous cell carcinoma. that as the cells acquire
to
9
simple
14
moderate dysplasia and 21 Our study indicated
a more malignant phenotype, there was a progressive increase in neu expression. It also suggested that neu may be involved in the development of oral cancers and that its evaluation in the early stages may assist in the diagnosis and management of oral
cancers.
oncogenes; Keywords: carcinoma; (HER-Z) ; oral chemistry
c-erbB-Z/neu immunohisto-
Correspondence to: Mien-Chie Hung, Dept. of Tumor Biology, M.D. Anderson Cancer Center, Box 79, 1515 Holcombe Blvd., Houston, TX 77030, USA.
0304-3835/92/$05.00 Printed and Published
0 1992 Elsevier Scientific Publishers in Ireland
C-erbB-Z/neu gene (also known as HER-2) was originally identified in neuroblastomas which developed in the offsprings of ethylnitrosourea injected pregnant rats [ 131. Subsequently, it was found to be homologous to the v-erbB oncogene and was cloned using v-erbB as a probe [7]. Neu is a 185kDa transmembrane protein with tyrosine kinase activity. It is highly homologous to but distinct from the epidermal growth factor receptor, EGFR. Activation of neu can occur by a single mutation in the transmembrane domain [2]. However, the pathogenesis of human malignancies appear to involve the overexpression of the normal neu gene product. In cases where there is neu without significant gene overexpression amplification, transcriptional and/or translational dysregulation may be responsible for neu overexpression [9]. Neu amplification has been detected in a variety of human primary tumors, including salivary adenocarcinoma [14], gastric carcinoma [4], colon carcinoma [3], lung carcinoma [12], ovarian carcinoma [17,24] and breast carcinoma [8,16]. These observations suggest that neu may be playing Ireland Ltd
216
an important role in the process of tumorigenesis. Furthermore, neu may also be involved in the processes of metastases. For instance, neu overexpression has been shown to correlate with lymph node metastases in breast and lung cancers [lo, 15,16,20]. Oral cancers as defined by the WHO (World Health Organization) is one of the 10 most frequent cancers in the world. The majority of these cases arose in the developing countries. Although oral cancer is relatively less common in the westernized countries, geographic heterogeneity is evident in certain areas. The role of cytokines in the growth and progression of squamous cell cancers has been studied recently. TGF-cr has been shown to have sequence homology to EGF and binds to EGFR. It appears to be involved in the growth and progression of these tumors [23]. TGF-fl and interleukin-1 have also been found to play important roles in the development of squamous cell cancers [ll], In addition, there is evidence for cross interaction between neu and EGFR [19] suggesting a role for neu in these tumors. This study was designed to investigate the role of neu in the development and progression of oral cancers. We analyzed the expression and distribution of neu protein in various tissues ranging from normal to progressively more malignant phenotypes. The evidence suggests that neu expression is associated with malignancy of the oral cavity. Materials
and Methods
Specimens All specimens were obtained from the Department of Oral Pathology of Shandong Medical University. These specimens were routinely fixed in formalin and embedded in paraffin, then serial sections with thickness of 5 pm were made. The diagnoses of epithelial dysplasia were made according to the 12 criteria put forth by WHO [22] and the criteria of Banoczy [l]. Eighty-six specimens were examined, including 9 cases of simple hyperplasia (6 leukoplakia, 3 lichen planus), 15 cases of mild dysplasia (12 leucoplakia, 2
lichen planus, 1 rhomboid glossitis), 14 cases of moderate dysplasia (11 leukoplakia, 1 lichen planus, 1 erythroplasia, 1 abnormal proliferation of submucosal fibers), 21 cases of invasive squamous cell carcinoma and 7 cases of normal oral mucosa. Reagent Monoclonal antibody c-neuMAb3 of c-erbB2 product was supplied by Oncogene Science Inc. Manhasset, NY, Biotinylated horse antimouse IgG and ABC kit were supplied by Vector Lab., Burlingame, CA. Method Immunoperoxidase staining technique was derived from Hsu’s ABC technique [5,6]. The procedure is as follows: (1) slides undergo deparaffination and hydration in gradient of alcohol; (2) blocked in 3% HsOs for 15 min, washed in TBS (0.05 M, pH 7.6); (3) digested in 0.05% trypsin for 20 min, washed in TBS; (4) treated in 10% horse serum for 20 min; (5) incubated with PAb3 (1: 100 or 10 pg/ml) for 60 min, then kept at 4OC overnight, washed in TBS; (6) incubated with biotinylated horse anti-mouse IgG (1:200) for 30 min, washed in TBS; (7) incubated with ABC reagent (1: 100) for 60 min, washed in TBS; (8) developed in 0.04% DAB-HsOs (40 mg of DAB in 100 ml of TBS, add 20 ~1 of 3% H202), washed in tap water; (9) stained with hematoxylin, dehydrated in alcohol, cleaned in xylol and mounted with resin. All procedures were performed at room temperature unless otherwise indicated. Negative controls in which TBS replaced the primary antibody were run with each batch of stains. A previously identified strongly staining tumor was used as a positive control. Intra and inter assay consistency was maintained by running these positive and negative controls with each batch of staining. Criteria of results The prepared slides were observed under optic microscope. It was assigned as positive for cells with brown granules and as negative for cells without brown granules.
217
.. t
, t. I*
1 . @,
A
oral cavity, (B) and (C) human oral Fig. 1. Immunohistochemical staining of ~185 neu in (A) normal human squamous cell carcinoma. Representative sections were stained with c-neu MAb3 monoclonal antibody at magnifications of x 100 (A and B) and x 400 (C).
218
Table I.
The relationship
Histol grades
between
histologic grades and the expression
Expression
of c-neu mAb3
of c-neu mAb3 in basal cell layer.
Rate of positive (%I
Normal oral mucosa Simple hyperplasia Mild dysplasia Moderate dysplasia Severe dysplasia Invasive carcinoma
n
+
-
7 9 15 14 20 21
0 0 2 10 16 21
7 9 13 4 4 0
Statistical method The data were analyzed using the test for a linear trend in proportions [18] and x2 test.
There was no significant difference between the basal cell layer and the prickle cell layer in the expression of neu. The increase in the positive rate of c-neuMAb3 expression, the intensity of c-neuMAb3 staining and the number of positive cells was noted to correlate with the progression of the oral tissue from dysplasia to carcinoma.
Results We did not detect expression of neu product in normal oral mucosa (Fig. la) and in simple hyperplasia (except in the prickle cell layer of a case of simple hyperplasia) . Neu product expression gradually becomes more evident and increases from mild to moderate and to severe dysplasia (linear trend test, P < 0.001). The increment is most remarkable and statistically significant in the transition from mild to moderate dysplasia (P < 0.05). Positive expression was also noted in all squamous cell carcinoma examined (Figs. lb, lc). The relationship between histologic grades of the oral specimens and the expression of neu as detected by c-neuMAb3 in the basal cell layer and in prickle cell layer was shown in Table I and Table II. Table II.
The relationship
Histol grades
between
0 0 13 71 80 100
Discussion Recent studies have indicated that malignant transformation may involve a multi-step process and that many if not all of these steps may implicate structural anomalies or regulatory dysfunction of oncogenes (or tumor suppressor genes). Oral cancers are more common in men than women. Smoking and alcoholic consumption are known risk factors for oral cancers. Moreover, certain papillomaviruses may also contribute to the development of oral cancers. Though the molecular mechanisms of action of these environmental
histologic grades and the expression
Expression
of c-neu mAb3
Rate of positive (%I
Normal oral mucosa Simple hyperplasia Mild dysplasia Moderate dysplasia Severe dysplasia Invasive carcinoma
n
+
7 9 15 14 20 21
0 1 6 11 17 21
of c-neu mAb3 in prickle cell layer.
7 8 9 3 3 0
0 11 40 79 85 100
219
risk factors are still largely unknown, recent studies have suggested that TGF-cr and IL-1 may be some of the involved mediators. Walker [Zl] made the original observation in 1989 that immunohistochemical staining of human breast cancer with polyclonal antiserum 21N against neu protein was able to distinguish tumor cells from normal breast epithelium. It was also demonstrated that staining was associated with positive nodal status and poorer nuclear grade thereby suggested the importance of immunohistochemistry in the diagnosis and prognosis of these cancers. This paper provides evidences for a potential relationship between neu protein overexpression and the malignant transformation of oral cancers. (1) The staining of simple hyperplasia and mild dysplasia of epithelium was similar to that of normal oral mucosa. It implied that there was no neu overexpression in these lesions. (2) The intensity of staining and the number of positive cells increased markedly from mild dysplasia to moderate and severe dysplasia. The phenomenon was even more pronounced in carcinomas. This implied neu overexpression in these later lesions. The degree of staining intensity and the number of positive cells discriminate moderate and severe dysplasia of the oral epithelium from normal and simple dysplastic mucosa. It may provide reliable and accurate criteria to diagnose precancerous lesions of the oral mucosa. (3) It was observed that cancer cells from all 21 cases of squamous cell carcinoma showed strong staining implying neu overexpression. Whether neu overexpression is the consequence of gene amplification or transcriptional/translational dysregulation needs further experimentation. The variation of staining by c-neuMAb3 in the carcinoma ceils suggests possible tumor heterogeneity. (4) A tendency towards increased neu product in progressively more advanced cancer was evident from our results. Assuming the degree of staining increased proportionally to the amount of neu product, the presence of neu may be involved in cell dedifferentiation.
(5) Finally, th e staining pattern of severe dysplasia of oral epithelium resembled that of carcinoma. The strong morphological similarity and the identical pattern of neu expression support the argument that moderate/severe dysplasia have a high risk of developing malignancy in precancerous lesions. As far as we know, this study provides the first evidence for a significant association between neu oncogene overexpression and oral cancer progression. By means of an immunohistochemical approach using monoclonal antibodies against neu protein, we demonstrated that there is overexpression of neu in more advanced stages of oral cancer. Whether overexpression of neu is due to gene amplification or due to transcriptional/translational dysregulation of a normal neu product is not known. That neu may be a discrete anomaly contributing to the multistep process of malignant transformation in oral cancer is a distinct possibility. This opens a novel avenue to the basic study and early diagnosis of oral cancer.
Acknowledgement
This work was partially supported Smokeless Tobacco Research Council No. 0287 to M.C. Hung. We would thank Terry Smith for her assistance statistical analysis.
by the Grant like to in our
References Banouy,
J. and Csiba, A. (1976)
Occurrence
dysplasia in oral leukoplakia.
Oral Surg.,
Bargmann,
and Weinberg,
C.I.,
Hung, M.C.
Multiple independent mutation
~185.
Cell, 45, 649-657.
D’Emilia,
altering
the
transmembrane
J., Bulovas, K., D’Ercole,
and Summerhayes,
erbB-2
gene
neoplastic
R.A.
(1986)
activations of the neu oncogene by a
point
J.G.
of epithelial
42, 766 - 774.
product
progression
I.C.
(1989)
of
K., Wolf, B., Steele, Expression of the c-
(~185)
at
in
colon.
the
domain
different
stages
Oncogene,
of
4,
1233-1239. Fukushige, S.-l., Matsubara, K.-l., Yoshida, M., Sasaki, M., Suzuki, T., Semba, K., Toyoshima. K. and Yamamoto, T. (1986) Localization of a novel v-erbBrelated
gene,
c-erbB-2,
on
chromosome
17
and
its
amplification in a gastric cancer cell line. Mol. Cell Biol., 6,
955-958.
220 5
6
7
8
9
10
11 12
13
14
15
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