- Email: [email protected]
neu Expression in Oral Squamous Cell Carcinoma
HER2/neu inMaxillofac Oral CancerSurg 2004;16:172-176. Asian J Oral ORIGINAL RESEARCH
HER2/neu Expression in Oral Squamous Cell Carcinoma Satoru Shintani, Yuuji Nakahara, Chunnan Li, Mariko Mihara, Koh-ichi Nakashiro, Hiroyuki Hamakawa Department of Oral and Maxillofacial Surgery, Ehime University School of Medicine, Ehime, Japan
Abstract Objective: To evaluate the expression of HER2/neu in oral squamous cell carcinoma with a view to determining the usefulness of molecular target therapy by anti-HER2 antibody. Patients and Methods: Oral squamous cell carcinoma cell lines and 69 clinical tumour samples were tested using enzyme-linked immunosorbent assay and immunohistochemistry. Positive and negative controls were utilised. Results: Expression of HER2/neu in oral squamous cell carcinoma cell lines was low. Overexpression was not observed in the clinical samples. Conclusion: Low prevalence of expression of HER2/neu in oral squamous cell carcinoma limits the likely utility of herceptin therapy Key words: HER2/neu, Immunohistochemistry, Squamous cell carcinoma
Introduction Since oral squamous cell carcinoma (OSCC) exhibits aggressive biologic behaviour, the development of new treatment modalities for the primary tumour, as well as for metastatic disease, remains a challenge. Studies of the molecular biology of cancer have demonstrated that activation of oncogenes plays an important role in the development and progression of several types of tumours. HER2/neu (also known as c-erbB-2) is a proto-oncogene located on the human chromosome 17 and encodes a 185-kD transmembrane glycoprotein with tyrosine kinase activity.1,2 This glycoprotein demonstrates extensive homology to the epidermal growth factor receptor.3 Amplification of HER2/neu oncogene or overexpression of its protein has been demonstrated in some malignant neoplasms, including oral cancer.4-7 If the OSCC exhibits overexpression of HER2/ neu, an attractive target for receptor-mediated immunotherapy could be considered. Herceptin (trastuzumab; Genentech, San Francisco, USA), a humanised monoclonal antibody directed against the Correspondence: Satoru Shintani, Department of Oral and Maxillofacial Surgery, Ehime University School of Medicine, 454 Shitsukawa, Shigenobucho, Onsen-gun, Ehime, 791-0295, Japan. Tel: (81 89) 960 5392; Fax: (81 89) 960 5396; E-mail: [email protected]
172
extracellular domain of the HER2/neu protein, has been studied in several large clinical multicentre trials as a first-line therapy for metastatic breast carcinomas that exhibit HER2/neu overexpression.8-10 Aberrant expression of HER2/neu has been frequently observed in OSCC but the reported results are controversial because of their wide range (between 0% and 88%).6,7,11-20 The aim of this study was to examine the expression of HER2/neu in both OSCC cell lines and human OSCC samples to validate or otherwise the controversial results of various studies and to determine whether herceptin could be considered as adjunctive therapy for local recurrence and metastasis in OSCC.
Patients and Methods Cell Cultures Ten OSCC cell lines (HSC2, HSC3, HSC4, SCC4, SCC9, SAS, Ca9-22, KB, Ho-1-N-1, Ho-1-U-1) were examined. Breast cancer cell lines SK-BR3 and MCF7 were also examined as positive and negative controls, respectively. All OSCC cells were obtained from the Japanese Collection Research Bioresources. SK-BR3 and MCF7 were donated from the cell resource centre for biomedical research, Tohoku University, Japan. The cell lines were maintained in Dulbecco’s Modified Eagle’s Medium/F12 medium Asian J Oral Maxillofac Surg Vol 16, No 3, 2004
Shintani, Nakahara, Li, et al
(Gibco-BRL, Gaithersburg, USA) supplemented with 10% heat-inactivated foetal bovine serum (Sigma, St Louis, USA), penicillin, and streptomycin at 37°C in 95% air/5% carbon dioxide. Tissue Samples Tissue samples of 69 OSCCs were obtained from previously untreated patients at the Department of Oral and Maxillofacial Surgery, Ehime University School of Medicine, Japan, from 1991 to 2001. The clinical data are summarised in Table 1. The median age of the 69 patients with OSCC was 65.3 years. Eleven patients had stage I disease, 26 had stage II, 14 had stage III, and 18 had stage IV according to the TNM Classfication.21 Measurement of HER2/neu by Enzymelinked Immunosorbent Assay The 10 OSCC cell lines were evaluated by enzymelinked immunosorbent assay (ELISA). HER2/neu expression was measured using the commercially available competitive ELISA kit from Oncogene Research Products (San Diego, USA). The amount of HER2/neu was extrapolated from the HER2 standard curve and expressed in ng/ml. Immunohistochemistry of Tissue Samples Immunohistochemical (IHC) studies were performed on the formalin-fixed paraffin-embedded materials with the standard, semiquantitative HercepTestTM kit (Dako, Japan), which uses primary rabbit antihuman polyclonal antibody against HER2/neu.22 The staining procedure was performed strictly according to the
manufacturer’s instructions. Supplied control slides within the kit were compared with the examined slides to verify the specificity of the IHC reactions. Assessment of the staining intensity was performed using the accepted clinical scoring criteria for breast cancer.23
Results HER2/neu Expression in Cell Lines A competitive ELISA assay was used to determine the expression level of OSCC cell lines, comparing it to that of SK-BR3 which overexpresses HER2/neu. As a negative control, MCF7 was selected as a cell with low expression of HER2/neu. As shown in Figure 1, MCF7 (negative control) expressed 0.5 ng HER2/neu/ml/5 x 106 cells and SK-BR3 (positive control) expressed 2.1 ng HER2/neu/ml/5 x 106 cells. OSCC cell lines expressed a range from 0.1 ng to 0.6 ng HER2/neu/ml/5 x 106 cells. Only Ho-1-U-1 expressed HER2/neu higher than MCF7. However, the level of expression was very low compared to SK-BR3 (Figure 1). HER2/neu Expression in Tissues No sample was observed to demonstrate moderate staining of the entire membrane in more than 10% of
0
0.5
1
1.5
2
2.5
(ng/ml)
MCF7 HSC2 HSC3 HSC4
Characteristic
Number of patients
SCC4
Age (range) [years]
65.3 ± 13.5 (40-92)
SCC9
Gender Male Female
45 24
Primary tumour sites Tongue Gingiva Floor of the mouth Buccal mucosa
26 26 12 5
Clinical stage I II III IV
11 26 14 18
SAS Ca-9-22 KB Ho-1-N-1 Ho-1-U-1 SK-BR3
Table 1. Characteristics of 69 patients with oral squamous cell carcinoma. Asian J Oral Maxillofac Surg Vol 16, No 3, 2004
Figure 1. HER2/neu expression levels evaluated by enzymelinked immunosorbent assay in oral squamous cell carcinoma cell lines.
173
HER2/neu in Oral Cancer
a
tumour cells (2+). HER2/neu overexpression was not identified in any of the cases we examined (Figure 2a). Weak and mostly partial membrane staining was demonstrated in 2 cases (2.9%) [Figure 2b]. Supplied positive control tissues were stained according to the manufacturer’s instructions and demonstrated strong expression (Figure 2c). There was no staining in sections stained with the negative control reagent.
Discussion
b
c
Figure 2. Immunohistochemical staining for the expression of HER2/neu in oral squamous cell carcinoma (OSCC). (a) Most OSCC tissue showed negative staining; (b) 2 cases (2.9%) with weak and mostly partial membrane staining were detected; and (c) the positive control (SK-BR3) was clearly staining strongly.
174
Gene amplification and overexpression of the epidermal growth factor receptor (EGFR) have been detected in OSCC, and the results of preclinical studies and early clinical trials suggest that targeting the EGFR could represent a significant contribution to cancer therapy.24 Several studies have described the possible role of EGFR family members in OSCC.6,7,11,15-20 The results of these studies varied greatly from no overexpression of HER2/neu,11,13,15,16 to 25%,20 46%,12 and more than 50%6,7,12-14,17-19 (Table 2). In the present study, HER2/neu overexpression was not observed. The various studies have revealed major discrepancies that could be explained by the type of antibody used to identify HER2/neu, the laboratory procedures, and the scoring criteria. A large number of different antibodies have been used to identify HER2/neu by IHC staining of OSCC tissue sections.7,12-14,16-19 In breast cancer research, some of these antibodies strongly stained tissues that demonstrated a concomitant high level of HER2/neu gene amplification, while at lower levels of amplification, their staining ability decreased.9,25 The antibody used in the present study had an 85% correlation level between its detection by IHC and gene amplification in breast cancer.9 The scoring criteria for HER2/neu staining by IHC is also important. In the present study, the 3-point scoring criteria were suitable for herceptin immunotherapy.9,26 In our series, only 2 cases (2.9%) were scored as 1+ (weak and mostly partial membrane staining) while the other cases were negative (0). With this 3-point scoring system, a score of 2+ (a weakmoderate complete membrane staining is observed in more than 10% of the tumour cells) and a score of 3+ (a strong complete membrane staining is observed in more than 10% of the tumour cells) would be Asian J Oral Maxillofac Surg Vol 16, No 3, 2004
Shintani, Nakahara, Li, et al
Study
Number of patients
Methods
Kearsley et al11
46
Southern Blot
0
No association with lymph node metastases or survival
Craven et al12
93
Immunohistochemistry
46
No association with lymph node metastases or survival
Field et al13
75
Immunohistochemistry
0
No association with lymph node metastases or survival
Hou et al
86
Immunohistochemistry
72
Significant association with degree of malignant progression
Irish and Bernstein15
47
Southern Blot
0
No association with lymph node metastases or survival
Rodrigo et al
59
Double-differential polymerase chain reaction
0
No association with lymph node metastases or survival
Werkmeister et al20
85
Immunohistochemistry
61
Significant association with lymph node metastases and decreased survival
Ibrahim et al18
16
Immunohistochemistry
88
Significant association between c-erbB-2 and stage
Xia et al
80
Immunohistochemistry
80
Significant association with lymph node metastases and decreased survival
Ibrahim et al19
26
Immunohistochemistry
58
No association with site, grade, lymph node metastases, stage
Xia et al7
47
Immunohistochemistry
67
Significant association with lymph node metastases lymph node metastases and decreased survival
Werkmeister et al17
110
Double-differential polymerase chain reaction
25
Significant association with decreased survival
14
16
6
Positive and amplified (%)
Findings
Table 2. Clinical studies of HER2/neu in head and neck squamous cell carcinoma.
candidates for herceptin immunotherapy. Of the 69 examined cases, no sample scored a positive result (2+). Thus, according to breast cancer criteria, OSCC did not express the level of HER2/neu suitable for clinical use of herceptin therapy. However, a competitive ELISA assay is available to evaluate the HER2 expression level of OSCC tissue. This method is more quantitative and may be useful to validate the results of this study.
References 1. Popescu NC, King CR, Kraus NH. Localization of the erbB-2 gene on normal and rearranged chromosomes 17 to bands of q 12-21.32. Genomics 1989;4:362-366. 2. Akiyama T, Sudo C, Ogawara H, Toyoshima K, Yamamoto T. The product of human c-erbB-2 gene: a 185-kDa glycoprotein with tyrosinekinase activity. Science 1986;232:1644-1646. 3. Yamamoto T, Ikawa S, Akiyama T. Similarity of protein encoded by the human c-erbB-2 gene to epidermal growth factor receptor. Nature 1986; 319:230-234. 4. Press MF, Cordon-Card C, Slamon DJ. Expression of the HER2/neu proto-oncogene in normal human adult and fetal tissues. Oncogene 1990;5:953-962. Asian J Oral Maxillofac Surg Vol 16, No 3, 2004
5. Venter DJ, Tuzi, NL Kumar S, Gullick WJ. Overexpression of the c-erbB-2 oncogene protein in human breast carcinomas; immunohistochemical assessment correlates with gene amplification. Lancet 1987;11:69-72. 6. Xia W, Lau YK, Zhang HZ, Liu AR, Li L, Kiyokawa N, Clayman GL, Katz RL, Hung MC. Strong correlation between c-erbB-2 overexpression and overall survival of patients with oral squamous cell carcinoma. Clin Cancer Res 1997;3:3-9. 7. Xia W, Lau YK, Zhang HZ, Xiao FY, Johnston DA, Liu AR, Li L, Katz RL, Hung MC. Combination of EGFR, HER-2/neu, and HER-3 is a stronger predictor for the outcome of oral squamous cell carcinoma than any individual family members. Clin Cancer Res 1999;5: 4164-4174. 8. Baselga J, Tripathy D, Mendelsohn J. Phase II study of weekly intravenous recombinant humanized anti-p185HER2 monoclonal antibody in patients with HER2/neu overexpressing metastatic breast cancer. J Clin Oncol 1996;14: 737-744. 9. Pegram MD, Lipton A, Hayes DF. Phase II study of receptor-enhanced chemosensitivity using recombinant humanized anti p185HER2/neu 175
HER2/neu in Oral Cancer
monoclonal antibody plus cisplatin in patients with HER2/neu overexpressing metastatic breast cancer refractory to chemotherapy treatment. J Clin Oncol 1998;16:2659-2671. 10. Cobleigh MA, Vogel CL, Tripathy D. Multinational study of the efficacy and safety of humanized anti-HER2 monoclonal antibody in women who have HER2 overexpressing metastatic breast cancer that has progressed after chemotherapy for metastatic disease. J Clin Oncol 1999;17:2639-2648. 11. Kearsley JH, Leonard JH, Walsh MD, Wright GR. A comparison of epidermal growth factor receptor (EGFR) and c-erbB-2 oncogene expression in head and neck squamous cell carcinomas. Pathology 1991;23:189-194. 12. Craven JM, Pavelic ZP, Stambrook PJ, Pavelic L, Gapany M, Kelley DJ. Expression of c-erbB2 gene in human head and neck carcinoma. Anticancer Res 1992;12:2273-2276. 13. Field JK, Spandidos DA, Yiagnisis M, Gosney JR, Papadimitriou K, Stell PM. c-erbB2 Expression in squamous cell carcinoma of the head and neck. Anticancer Res 1992;12:613-620. 14. Hou L, Shi D, Tu SM, Zhang HZ, Hung MC, Ling D. Oral cancer progression and c-erbB-2/neu proto-oncogene expression. Cancer Lett 1992;65: 215-220. 15. Irish J, Bernstein A. Oncogenes in head and neck cancer. Laryngoscope 1993;103:42-52. 16. Rodrigo JP, Ramos S, Lazo PS, Alvarez I, Suarez C. Amplification of ERBB oncogenes in squamous cell carcinomas of the head and neck. Eur J Cancer Part A 1996;32:2004-2010. 17. Werkmeister R, Brandt B, Joos U. The erbB oncogenes as prognostic markers in oral squamous cell carcinomas. Am J Surg 1996;172:681-683. 18. Ibrahim SO, Vasstrand EN, Liavaag PG, Johannessen AC, Lillehaug JR. Expression of c-erbB proto-oncogene family members in
176
squamous cell carcinoma of the head and neck. Anticancer Res 1997;17:4539-4546. 19. Ibrahim SO, Lillehaug JR, Johannessen AC, Liavaag PG, Nilsen R, Vasstrand EN. Expression of biomarkers (p53, transforming growth factor alpha, epidermal growth factor receptor, c-erbB2/neu and the proliferative cell nuclear antigen) in oropharyngeal squamous cell carcinomas. Oral Oncol 1999;35:302-313. 20. Werkmeister R, Brandt B, Joos U. Clinical relevance of erbB-1 and-2 oncogenes in oral carcinomas. Oral Oncol 2000;36:100-105. 21. Sobin LH, Fleming ID. TNM Classification of malignant tumors. 5th ed. Union Internationale Contre le Cancer and the American Joint Committee on Cancer. Cancer 1997;80:1803-1804. 22. Espinoza F, Anguiano A. The HercepTest assay: another perspective. J Clin Oncol 1999;17: 2293-2294. 23. Mitchell MS, Press MF. The role of immunohistochemistry and fluorescence in situ hybridization for HER2/neu in assessing the prognosis of breast cancer. Semin Oncol 1999;26:108-116. 24. Ciardiello F, Tortora G. A novel approach in the treatment of cancer: targeting the epidermal growth factor receptor. Clin Cancer Res 2001; 7:2958-2970. 25. Press MF, Hung G, Godolphin W, Slamon DJ. Sensitivity of HER2/neu antibodies in archival tissue samples: potential source of error in immunohistochemical studies of oncogene expression. Cancer Res 1994;54:2771-2777. 26. Cobleigh MA, Vogel CL, Tripathy D. Multinational study of the efficacy and safety of humanized anti-HER2 monoclonal antibody in women who have HER2 overexpressing metastatic breast cancer that has progressed after chemotherapy for metastatic disease. J Clin Oncol 1999;17:2639-2648.
Asian J Oral Maxillofac Surg Vol 16, No 3, 2004
HER2/neu Expression in Oral Squamous Cell Carcinoma Satoru Shintani, Yuuji Nakahara, Chunnan Li, Mariko Mihara, Koh-ichi Nakashiro, Hiroyuki Hamakawa Department of Oral and Maxillofacial Surgery, Ehime University School of Medicine, Ehime, Japan
Abstract Objective: To evaluate the expression of HER2/neu in oral squamous cell carcinoma with a view to determining the usefulness of molecular target therapy by anti-HER2 antibody. Patients and Methods: Oral squamous cell carcinoma cell lines and 69 clinical tumour samples were tested using enzyme-linked immunosorbent assay and immunohistochemistry. Positive and negative controls were utilised. Results: Expression of HER2/neu in oral squamous cell carcinoma cell lines was low. Overexpression was not observed in the clinical samples. Conclusion: Low prevalence of expression of HER2/neu in oral squamous cell carcinoma limits the likely utility of herceptin therapy Key words: HER2/neu, Immunohistochemistry, Squamous cell carcinoma
Introduction Since oral squamous cell carcinoma (OSCC) exhibits aggressive biologic behaviour, the development of new treatment modalities for the primary tumour, as well as for metastatic disease, remains a challenge. Studies of the molecular biology of cancer have demonstrated that activation of oncogenes plays an important role in the development and progression of several types of tumours. HER2/neu (also known as c-erbB-2) is a proto-oncogene located on the human chromosome 17 and encodes a 185-kD transmembrane glycoprotein with tyrosine kinase activity.1,2 This glycoprotein demonstrates extensive homology to the epidermal growth factor receptor.3 Amplification of HER2/neu oncogene or overexpression of its protein has been demonstrated in some malignant neoplasms, including oral cancer.4-7 If the OSCC exhibits overexpression of HER2/ neu, an attractive target for receptor-mediated immunotherapy could be considered. Herceptin (trastuzumab; Genentech, San Francisco, USA), a humanised monoclonal antibody directed against the Correspondence: Satoru Shintani, Department of Oral and Maxillofacial Surgery, Ehime University School of Medicine, 454 Shitsukawa, Shigenobucho, Onsen-gun, Ehime, 791-0295, Japan. Tel: (81 89) 960 5392; Fax: (81 89) 960 5396; E-mail: [email protected]
172
extracellular domain of the HER2/neu protein, has been studied in several large clinical multicentre trials as a first-line therapy for metastatic breast carcinomas that exhibit HER2/neu overexpression.8-10 Aberrant expression of HER2/neu has been frequently observed in OSCC but the reported results are controversial because of their wide range (between 0% and 88%).6,7,11-20 The aim of this study was to examine the expression of HER2/neu in both OSCC cell lines and human OSCC samples to validate or otherwise the controversial results of various studies and to determine whether herceptin could be considered as adjunctive therapy for local recurrence and metastasis in OSCC.
Patients and Methods Cell Cultures Ten OSCC cell lines (HSC2, HSC3, HSC4, SCC4, SCC9, SAS, Ca9-22, KB, Ho-1-N-1, Ho-1-U-1) were examined. Breast cancer cell lines SK-BR3 and MCF7 were also examined as positive and negative controls, respectively. All OSCC cells were obtained from the Japanese Collection Research Bioresources. SK-BR3 and MCF7 were donated from the cell resource centre for biomedical research, Tohoku University, Japan. The cell lines were maintained in Dulbecco’s Modified Eagle’s Medium/F12 medium Asian J Oral Maxillofac Surg Vol 16, No 3, 2004
Shintani, Nakahara, Li, et al
(Gibco-BRL, Gaithersburg, USA) supplemented with 10% heat-inactivated foetal bovine serum (Sigma, St Louis, USA), penicillin, and streptomycin at 37°C in 95% air/5% carbon dioxide. Tissue Samples Tissue samples of 69 OSCCs were obtained from previously untreated patients at the Department of Oral and Maxillofacial Surgery, Ehime University School of Medicine, Japan, from 1991 to 2001. The clinical data are summarised in Table 1. The median age of the 69 patients with OSCC was 65.3 years. Eleven patients had stage I disease, 26 had stage II, 14 had stage III, and 18 had stage IV according to the TNM Classfication.21 Measurement of HER2/neu by Enzymelinked Immunosorbent Assay The 10 OSCC cell lines were evaluated by enzymelinked immunosorbent assay (ELISA). HER2/neu expression was measured using the commercially available competitive ELISA kit from Oncogene Research Products (San Diego, USA). The amount of HER2/neu was extrapolated from the HER2 standard curve and expressed in ng/ml. Immunohistochemistry of Tissue Samples Immunohistochemical (IHC) studies were performed on the formalin-fixed paraffin-embedded materials with the standard, semiquantitative HercepTestTM kit (Dako, Japan), which uses primary rabbit antihuman polyclonal antibody against HER2/neu.22 The staining procedure was performed strictly according to the
manufacturer’s instructions. Supplied control slides within the kit were compared with the examined slides to verify the specificity of the IHC reactions. Assessment of the staining intensity was performed using the accepted clinical scoring criteria for breast cancer.23
Results HER2/neu Expression in Cell Lines A competitive ELISA assay was used to determine the expression level of OSCC cell lines, comparing it to that of SK-BR3 which overexpresses HER2/neu. As a negative control, MCF7 was selected as a cell with low expression of HER2/neu. As shown in Figure 1, MCF7 (negative control) expressed 0.5 ng HER2/neu/ml/5 x 106 cells and SK-BR3 (positive control) expressed 2.1 ng HER2/neu/ml/5 x 106 cells. OSCC cell lines expressed a range from 0.1 ng to 0.6 ng HER2/neu/ml/5 x 106 cells. Only Ho-1-U-1 expressed HER2/neu higher than MCF7. However, the level of expression was very low compared to SK-BR3 (Figure 1). HER2/neu Expression in Tissues No sample was observed to demonstrate moderate staining of the entire membrane in more than 10% of
0
0.5
1
1.5
2
2.5
(ng/ml)
MCF7 HSC2 HSC3 HSC4
Characteristic
Number of patients
SCC4
Age (range) [years]
65.3 ± 13.5 (40-92)
SCC9
Gender Male Female
45 24
Primary tumour sites Tongue Gingiva Floor of the mouth Buccal mucosa
26 26 12 5
Clinical stage I II III IV
11 26 14 18
SAS Ca-9-22 KB Ho-1-N-1 Ho-1-U-1 SK-BR3
Table 1. Characteristics of 69 patients with oral squamous cell carcinoma. Asian J Oral Maxillofac Surg Vol 16, No 3, 2004
Figure 1. HER2/neu expression levels evaluated by enzymelinked immunosorbent assay in oral squamous cell carcinoma cell lines.
173
HER2/neu in Oral Cancer
a
tumour cells (2+). HER2/neu overexpression was not identified in any of the cases we examined (Figure 2a). Weak and mostly partial membrane staining was demonstrated in 2 cases (2.9%) [Figure 2b]. Supplied positive control tissues were stained according to the manufacturer’s instructions and demonstrated strong expression (Figure 2c). There was no staining in sections stained with the negative control reagent.
Discussion
b
c
Figure 2. Immunohistochemical staining for the expression of HER2/neu in oral squamous cell carcinoma (OSCC). (a) Most OSCC tissue showed negative staining; (b) 2 cases (2.9%) with weak and mostly partial membrane staining were detected; and (c) the positive control (SK-BR3) was clearly staining strongly.
174
Gene amplification and overexpression of the epidermal growth factor receptor (EGFR) have been detected in OSCC, and the results of preclinical studies and early clinical trials suggest that targeting the EGFR could represent a significant contribution to cancer therapy.24 Several studies have described the possible role of EGFR family members in OSCC.6,7,11,15-20 The results of these studies varied greatly from no overexpression of HER2/neu,11,13,15,16 to 25%,20 46%,12 and more than 50%6,7,12-14,17-19 (Table 2). In the present study, HER2/neu overexpression was not observed. The various studies have revealed major discrepancies that could be explained by the type of antibody used to identify HER2/neu, the laboratory procedures, and the scoring criteria. A large number of different antibodies have been used to identify HER2/neu by IHC staining of OSCC tissue sections.7,12-14,16-19 In breast cancer research, some of these antibodies strongly stained tissues that demonstrated a concomitant high level of HER2/neu gene amplification, while at lower levels of amplification, their staining ability decreased.9,25 The antibody used in the present study had an 85% correlation level between its detection by IHC and gene amplification in breast cancer.9 The scoring criteria for HER2/neu staining by IHC is also important. In the present study, the 3-point scoring criteria were suitable for herceptin immunotherapy.9,26 In our series, only 2 cases (2.9%) were scored as 1+ (weak and mostly partial membrane staining) while the other cases were negative (0). With this 3-point scoring system, a score of 2+ (a weakmoderate complete membrane staining is observed in more than 10% of the tumour cells) and a score of 3+ (a strong complete membrane staining is observed in more than 10% of the tumour cells) would be Asian J Oral Maxillofac Surg Vol 16, No 3, 2004
Shintani, Nakahara, Li, et al
Study
Number of patients
Methods
Kearsley et al11
46
Southern Blot
0
No association with lymph node metastases or survival
Craven et al12
93
Immunohistochemistry
46
No association with lymph node metastases or survival
Field et al13
75
Immunohistochemistry
0
No association with lymph node metastases or survival
Hou et al
86
Immunohistochemistry
72
Significant association with degree of malignant progression
Irish and Bernstein15
47
Southern Blot
0
No association with lymph node metastases or survival
Rodrigo et al
59
Double-differential polymerase chain reaction
0
No association with lymph node metastases or survival
Werkmeister et al20
85
Immunohistochemistry
61
Significant association with lymph node metastases and decreased survival
Ibrahim et al18
16
Immunohistochemistry
88
Significant association between c-erbB-2 and stage
Xia et al
80
Immunohistochemistry
80
Significant association with lymph node metastases and decreased survival
Ibrahim et al19
26
Immunohistochemistry
58
No association with site, grade, lymph node metastases, stage
Xia et al7
47
Immunohistochemistry
67
Significant association with lymph node metastases lymph node metastases and decreased survival
Werkmeister et al17
110
Double-differential polymerase chain reaction
25
Significant association with decreased survival
14
16
6
Positive and amplified (%)
Findings
Table 2. Clinical studies of HER2/neu in head and neck squamous cell carcinoma.
candidates for herceptin immunotherapy. Of the 69 examined cases, no sample scored a positive result (2+). Thus, according to breast cancer criteria, OSCC did not express the level of HER2/neu suitable for clinical use of herceptin therapy. However, a competitive ELISA assay is available to evaluate the HER2 expression level of OSCC tissue. This method is more quantitative and may be useful to validate the results of this study.
References 1. Popescu NC, King CR, Kraus NH. Localization of the erbB-2 gene on normal and rearranged chromosomes 17 to bands of q 12-21.32. Genomics 1989;4:362-366. 2. Akiyama T, Sudo C, Ogawara H, Toyoshima K, Yamamoto T. The product of human c-erbB-2 gene: a 185-kDa glycoprotein with tyrosinekinase activity. Science 1986;232:1644-1646. 3. Yamamoto T, Ikawa S, Akiyama T. Similarity of protein encoded by the human c-erbB-2 gene to epidermal growth factor receptor. Nature 1986; 319:230-234. 4. Press MF, Cordon-Card C, Slamon DJ. Expression of the HER2/neu proto-oncogene in normal human adult and fetal tissues. Oncogene 1990;5:953-962. Asian J Oral Maxillofac Surg Vol 16, No 3, 2004
5. Venter DJ, Tuzi, NL Kumar S, Gullick WJ. Overexpression of the c-erbB-2 oncogene protein in human breast carcinomas; immunohistochemical assessment correlates with gene amplification. Lancet 1987;11:69-72. 6. Xia W, Lau YK, Zhang HZ, Liu AR, Li L, Kiyokawa N, Clayman GL, Katz RL, Hung MC. Strong correlation between c-erbB-2 overexpression and overall survival of patients with oral squamous cell carcinoma. Clin Cancer Res 1997;3:3-9. 7. Xia W, Lau YK, Zhang HZ, Xiao FY, Johnston DA, Liu AR, Li L, Katz RL, Hung MC. Combination of EGFR, HER-2/neu, and HER-3 is a stronger predictor for the outcome of oral squamous cell carcinoma than any individual family members. Clin Cancer Res 1999;5: 4164-4174. 8. Baselga J, Tripathy D, Mendelsohn J. Phase II study of weekly intravenous recombinant humanized anti-p185HER2 monoclonal antibody in patients with HER2/neu overexpressing metastatic breast cancer. J Clin Oncol 1996;14: 737-744. 9. Pegram MD, Lipton A, Hayes DF. Phase II study of receptor-enhanced chemosensitivity using recombinant humanized anti p185HER2/neu 175
HER2/neu in Oral Cancer
monoclonal antibody plus cisplatin in patients with HER2/neu overexpressing metastatic breast cancer refractory to chemotherapy treatment. J Clin Oncol 1998;16:2659-2671. 10. Cobleigh MA, Vogel CL, Tripathy D. Multinational study of the efficacy and safety of humanized anti-HER2 monoclonal antibody in women who have HER2 overexpressing metastatic breast cancer that has progressed after chemotherapy for metastatic disease. J Clin Oncol 1999;17:2639-2648. 11. Kearsley JH, Leonard JH, Walsh MD, Wright GR. A comparison of epidermal growth factor receptor (EGFR) and c-erbB-2 oncogene expression in head and neck squamous cell carcinomas. Pathology 1991;23:189-194. 12. Craven JM, Pavelic ZP, Stambrook PJ, Pavelic L, Gapany M, Kelley DJ. Expression of c-erbB2 gene in human head and neck carcinoma. Anticancer Res 1992;12:2273-2276. 13. Field JK, Spandidos DA, Yiagnisis M, Gosney JR, Papadimitriou K, Stell PM. c-erbB2 Expression in squamous cell carcinoma of the head and neck. Anticancer Res 1992;12:613-620. 14. Hou L, Shi D, Tu SM, Zhang HZ, Hung MC, Ling D. Oral cancer progression and c-erbB-2/neu proto-oncogene expression. Cancer Lett 1992;65: 215-220. 15. Irish J, Bernstein A. Oncogenes in head and neck cancer. Laryngoscope 1993;103:42-52. 16. Rodrigo JP, Ramos S, Lazo PS, Alvarez I, Suarez C. Amplification of ERBB oncogenes in squamous cell carcinomas of the head and neck. Eur J Cancer Part A 1996;32:2004-2010. 17. Werkmeister R, Brandt B, Joos U. The erbB oncogenes as prognostic markers in oral squamous cell carcinomas. Am J Surg 1996;172:681-683. 18. Ibrahim SO, Vasstrand EN, Liavaag PG, Johannessen AC, Lillehaug JR. Expression of c-erbB proto-oncogene family members in
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Asian J Oral Maxillofac Surg Vol 16, No 3, 2004