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Estimation of Serum Tumour Necrosis Factor-α and Correlation to Tumour Markers in Patients with Oral Squamous Cell Carcinoma
Tumour Necrosis Factor-α in Oral Squamous Cell Carcinoma ORIGINAL RESEARCH
Asian J Oral Maxillofac Surg 2002;14:148-154.
Estimation of Serum Tumour Necrosis Factor-α and Correlation to Tumour Markers in Patients with Oral Squamous Cell Carcinoma Hideo Kurokawa,1 Shinobu Takeda,1 Yoshihiro Yamashita,1 Takashi Nakamura,1 Tomoyuki Murata,1 Tetsu Takahashi,1 Hiroshi Fukuyama,2 Min Zhang,2 Hiroaki Ishibashi3 1 Second Department of Oral and Maxillofacial Surgery, Kyushu Dental College, Kitakyushu, Japan 2 Department of Oral Pathology, Kyushu Dental College, Kitakyushu, Japan 3 Second Department of Oral and Maxillofacial Surgery, School of Dentistry, Kyushu University, Fukuoka, Japan
Abstract
Objectives: Estimation of serum tumour necrosis factor-α levels and correlation with tumour markers for the prognosis of oral squamous cell carcinoma before treatment. Patients and Methods: Serum levels of tumour necrosis factor-α and 3 other tumour markers — carcinoembryonic antigen, squamous cell carcinoma antigen, and immunosuppressive acidic protein — were simultaneously measured in 38 patients with oral squamous cell carcinoma and in 36 healthy controls. Results: The mean serum concentration of tumour necrosis factor-α for patients with oral squamous cell carcinoma (14.1 ± 13.2 pg/mL) was significantly higher than that of healthy controls (5.2 ± 3.1 pg/mL) [p < 0.05]. The mean serum concentration of tumour necrosis factor-α was lower for patients who were pathological node-positive than for pathological node-negative patients. A significant correlation was observed between the serum tumour necrosis factor-α levels and pathological parameters. The mean serum concentration of tumour necrosis factor-α was also significantly higher for patients who had a good clinical course (16.7 ± 18.6 pg/mL) than for those with a poor clinical course (9.6 ± 5.9 pg/mL) [p < 0.05]. The survival curve revealed a better prognosis for serum tumour necrosis factor-α-positive patients than for serum tumour necrosis factor-α -negative patients. A significant correlation was observed between the serum tumour necrosis factor-α levels and the tumour markers, squamous cell carcinoma antigen and immunosuppressive acidic protein (p < 0.05). Conclusion: An evaluation of serum tumour necrosis factor-α level in patients with oral squamous cell carcinoma appears to be a valuable screening tool for the prognosis and treatment of patients with this cancer. Key Words: Tumour necrosis factors, Carcinoma, squamous cell, Tumour markers, biological, Prognosis
Introduction Tumour necrosis factor-α (TNF-α), a product of activated macrophages, is an important mediator in cytoprotection.1 TNF-α has been reported to exert an anti-tumour effect through both an indirect mechanism and via the activation of such immunologically competent cells as neutrophils and macrophages.2,3 Correspondence: Hideo Kurokawa, DDS, PhD, Second Department of Oral and Maxillofacial Surgery, Kyushu Dental College, 2-6-1 Manazuru, Kokurakita-ku, Kitakyushu City, 803-8580, Japan. Tel: (81 93) 582 1131, Fax: (81 93) 582 1139 E-mail: [email protected]
148
Estimates of the serum TNF-a level in cancer patients prior to surgery vary greatly,4 but there have been few studies on the usefulness of such determinations in establishing the diagnosis and prognosis of oral squamous cell carcinoma (O-SCC). Other tumour markers that have been used for patients with O-SCC include carcinoembryonic antigen (CEA),5,6 squamous cell carcinoma antigen (SCCA),5,7 immunosuppressive acidic protein (IAP),5 glutathione S-transferase-π (GST-π),8 dipeptidylpeptidase (DPP),9 and fragment of cytokeratin subunit 19 (Cyfra).10,11 A combination assay of CEA, Asian J Oral Maxillofac Surg Vol 14, No 3, 2002
Kurokawa, Takeda, Yamashita, et al
SCCA, and IAP has been reported to be useful for screening patients with O-SCC. 12 This study evaluated the use of preoperative serum TNF-a levels as a means of establishing both the diagnosis and postoperative prognosis of O-SCC. In addition, the relationships between the serum TNF-a level and that of the tumour markers CEA, SCCA, and IAP was investigated.
Patients and Methods Thirty eight consecutive patients with O-SCC, who were treated at Kyushu Dental College in Japan between 1989 and 1992, were entered into the study. The patients ages ranged from 34 to 89 years (mean, 63.8 years) — 23 were men and 15 were women. The anatomic sites of O-SCC were the tongue (n = 15), mandibular gingiva (n = 11), maxillary gingiva (n = 9), floor of the mouth (n = 2), and buccal mucosa (n = 1). Preoperatively, all patients were categorised according to the International Union Against Cancer (UICC) TNM classification (1987),13 as follows: • 9 patients had T1 • 11 patients had T2 • 7 patients had T3 • 11 patients had T4 • 26 patients had N0 • 12 patients had N1 or N2 • all patients had M0. According to the stage of disease, 8 patients demonstrated stage I disease, 9 had stage II disease, 8 had stage III disease, and 13 had stage IV disease. All patients received combined preoperative chemotherapy with cis-platinum (160 mg), peplomycin (50 mg), and tegafur uracil (300 to 600 mg/ day). The patients were separated into 2 groups after treatment. Sixteen patients had a good clinical course and remained completely free of the disease for more than 5 years after treatment of the primary tumour. The poor-clinical course group consisted of 22 patients who had recurrent tumours at the primary site, or died due to local recurrence at the primary site and either regional lymph node metastasis or distant metastasis. The control group comprised 36 healthy individuals. The mean age was 61.3 years (range, 22 to 82 years) — 20 were male and 16 were female. Asian J Oral Maxillofac Surg Vol 14, No 3, 2002
Blood specimens were taken after informed consent was obtained. The sera were centrifuged for 20 minutes at 2000 rpm, and then stored at -80°C until analysis. The serum TNF-α levels were measured with an enzyme-linked immunosorbent assay (ELISA) using a highly sensitive TNF assay kit (Tore Fuji Bionics, Tokyo, Japan). One monoclonal antibody was immobilised in each well of a 96-well microplate. After the plates were washed with phosphate-buffered saline (PBS), 200 μL of standard and test samples were allowed to react with this antibody and were incubated for 2 hours at room temperature. After 3 washes, a second monoclonal antibody was added to each well and incubated for 2 hours at room temperature. The samples were washed again and 200 μL of substrate was then added and incubated for 30 minutes at room temperature in the dark. The absorbance of the product was measured at 490 nm. The detection threshold of the serum TNF-α level was 1.9 pg/mL. Based on the receiver operating characteristic (ROC) curves for the serum TNF-α levels, a threshold of 6.5 pg/ml was chosen as the upper limit of the normal (ULN) — values above this threshold were defined as TNF-αpositive. The serum levels of CEA were determined by radioimmunoassay (CEA Roche Z Gel Kit, Roche Tokyo, Japan) originally developed by Thomson et al.14 The ULN was set at 2.5 ng/mL based on the findings of previous reports.5,6 The sensitivity of the test was 0.5 ng/mL. For the measurement of serum SCCA, a doubleantibody radioimmunoassay kit was used (SCC RIA BEAD, Dainabot Co, Tokyo, Japan). The ULN was considered to be 2.0 ng/mL.15 The sensitivity of the test was 0.7 ng/mL. IAP was quantitatively assayed in the sera by single radial immunodiffusion, as described by Mancini et al,16 using commercially available agarose plates. These plates, which contain rabbit anti-IAP, were bought from Kayaku Antibiotics Research Co. (Tokyo, Japan). The ULN was considered to be 500 μg/mL.17 149
Tumour Necrosis Factor-α in Oral Squamous Cell Carcinoma
Data were summarised as the mean activity plus or minus the standard deviation (A ± SD). A statistical analysis was performed using the arithmetic mean, the Mann-Whitney U test, and the chi-squared test. The survival rates were calculated by the KaplanMeier method, while the statistical difference was calculated by the log-rank test. A value of p < 0.05 was considered to be statistically significant.
Results Comparison of Serum Tumour Necrosis Factor- α Levels Between Patients and Controls The mean serum concentration of TNF-α was significantly higher in untreated patients with O-SCC than in healthy controls (p < 0.05). Twenty five of 38 patients (65.8%) were found to have elevated serum levels (Figure 1). Relationship Between Serum Tumour Necrosis Factor-α and Clinical Parameters The mean serum TNF-α level was 14.9 ± 10.3 pg/mL for T1/T2 (n = 20) and 10.5 ± 5.9 pg/mL for T3/T4 150
(pg/ml) 100 p < 0.05
TNF-α level
A histological examination was performed on the hematoxylin and eosin-stained sections prepared from either specimens biopsied before treatment or from surgical specimens. The histological malignancy grading system was determined according to Anneroth and Batsakis18 after evaluating the degree of keratinisation, nuclear polymorphism, number of mitosis, pattern of invasion, stage of invasion, and lymphoplasmatic infiltration. Each of these features was graded from 1 to 4 according to the definitions, giving a total malignancy score ranging from 6 to 24. The tumours were divided into 3 groups. The lowgrade malignancy group had a total malignancy score of <12, the moderate-grade malignancy group score ranged from 13 to 16, and the high-grade malignancy group had ≥17 points. All grading was performed independently by 2 pathologists. The relationships between the serum TNF-α levels, clinical (including T [T1/T2 and T3/T4] and pathological node [pN+ and pN-] categories) and stage classification (stages 1/2 and stages 3/4), and pathological parameters (including degree of keratinisation, lymphoplasmatic infiltration, mode of invasion,19 and histological malignancy) were evaluated.
Positive Negative
50
14.1±13.2 6.5
Positive rate
5.2±3.1
Healthy controls (n = 36) 11.1% (4 patients)
O-SCC patients (n = 38) 65.8% (25 patients)
p < 0.05
Figure 1. Comparison of serum tumour necrosis factor-α (TNF-α) levels among patients with oral squamous cell carcinoma (O-SCC) and healthy controls.
(n = 18) — the difference was not significant (Table 1). However, serum TNF-α levels were significantly lower in the patients who had metastatic lymph nodes (10.9 ± 9.8 pg/mL; n = 14) than in patients without metastatic lymph nodes (16.1 ± 16.2 pg/mL; n = 24) [p < 0.05; Table 1]. When the TNF-α level was analysed according to the UICC staging system,13 no significant differences were observed between the various stages (Table 1). Relationship Between Tumour Necrosis Factor- α and Pathological Parameters The mean serum TNF-α levels relative to the degree of keratinisation was 24.7 ± 38.4 pg/mL for the patients with a high degree of keratinisation (n = 24) Clinical parameters
Tumour necrosis factor-α level (pg/mL)
p Value
Tumour T1/T2 (n = 20) T3/T4 (n = 18)
14.9 ± 10.3 10.5 ± 5.9
NS
Node pN- (n = 24) pN+ (n = 14)
16.1 ± 16.2 10.9 ± 9.8
p < 0.05
Stage I/II (n = 17) III/IV (n = 21)
14.6 ± 9.5 13.1 ± 10.1
NS
Table 1. Relationship between serum tumour necrosis factor-α levels and the clinical parameters.
Asian J Oral Maxillofac Surg Vol 14, No 3, 2002
Kurokawa, Takeda, Yamashita, et al Tumour necrosis factor-α level (pg/mL)
p Value
Degree of keratinisation High (n = 24) Moderate (n = 14)
24.7 ± 38.4 8.5 ± 5.1
< 0.05
Lymphoplasmatic infiltration Slight, moderate (n = 24) Marked (n = 14)
10.5 ± 7.9 16.4 ± 19.4
< 0.05
Mode of invasion Grade 1 to 3 (n = 27) Grade 4C to 4D (n = 11)
18.4 ± 10.3 9.2 ± 6.6
< 0.05
Histological malignancy Low grade (n = 15) Moderate, high (n = 23)
20.1 ± 18.2 9.1 ± 6.4
< 0.05
Table 2. Relationship between serum tumour necrosis factor-α levels and pathological parameters.
and 8.5 ± 5.1 pg/mL for patients with a moderate degree of keratinisation (n = 14) — a significant difference between the 2 groups (p < 0.05). The mean serum TNF-α level relative to the degree of lymphoplasmatic infiltration was 10.5 ± 7.9 pg/mL for the slightly and moderately infiltrated patients (n = 24) and 16.4 ± 19.4 pg/mL for the markedly infiltrated group (n = 14), with a significant difference between them (p < 0.05). Regarding the mode of invasion, the mean serum TNF-α levels of the grades 1 to 3 groups (n = 27) were higher than for those with grades 4C (cord like pattern)/4D (diffuse pattern) [n = 11]. In addition, the mean serum TNF-α levels in patients with low histological malignancy were higher than those of the moderate and high histological malignancy group (Table 2). Relationship Between Tumour Necrosis Factor- α Levels and 5-Year Survival The mean serum concentration of TNF-α was significantly higher in patients with a good clinical course (16.7 ± 18.6 pg/mL, n = 16) than in patients with a poor clinical course (9.6 ± 5.9 pg/mL, n = 22) [p < 0.05]. The 5-year cumulative survival rate of the TNF-αpositive group was higher (80.4%) than that of the TNF-α-negative group (38.5%) [p < 0.05; Figure 2]. Relationship Between Tumour Necrosis Factor- α and Tumour Markers The relationship between serum TNF-α level and the other tumour markers is shown in Table 3. Although Asian J Oral Maxillofac Surg Vol 14, No 3, 2002
100
TNF-αpositive 80.4%
80
Survival rate (%)
Pathological parameters
60 TNF-αnegative 38.5%
40
20 p < 0.05 0
10
20
30
40
50
60
Time (months)
Figure 2. Overall actuarial survival curves by tumour necrosis factor-α (TNF-α).
no significant difference was seen between the CEA-negative group and the CEA-positive group with respect to the serum TNF-α level, the serum TNF-α level in the SCCA-negative group was significantly lower than in the SCCA-positive group (p < 0.05). The serum TNF-α level in the IAPnegative group was also significantly higher than in the IAP-positive group (p < 0.05).
Discussion TNF-α is a polypeptide cytokine produced by stimulated macrophages and lymphocytes.1 TNF-α was initially thought to be useful as an anticancer drug, free from side effects, but it was later shown to be identical to cachectin, and is now known to have a variety of biological activities.20 Many authors have Tumour marker
Tumour necrosis p Value factor-α level (pg/mL)
Carcinoembryonic antigen Positive (n = 16) Negative (n = 17)
10.1 ±6.1 14.4 ± 17.3
NS
Squamous cell carcinoma antigen Positive (n = 12) Negative (n = 21)
7.8 ± 5.5 15.0 ±15.4
< 0.05
Immunosuppressive acidic protein Positive (n = 12) 17.4 ± 18.5 Negative (n = 21) 8.9 ± 6.0
< 0.05
Table 3. Relationship between serum tumour necrosis factor-α levels and other tumour markers (carcinoembryonic antigen, squamous cell carcinoma antigen, and immunosuppressive acidic protein).
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Tumour Necrosis Factor-α in Oral Squamous Cell Carcinoma
reported that TNF-α acts directly against cancer cells by exerting antitumour effects through an indirect mechanism and via the activation of immunologically competent cells such as neutrophils and macrophages.2,3 A deficiency in TNF-α production by monocytes may therefore result in a susceptibility to infection, malignancy, and other physiological abnormalities.21 However, the mechanism of action of TNF-α in humans remains controversial. Many investigators have also found that the level of biologically active or immunoreactive TNF-α is associated with outcome in patients with malignant tumours,22,23 although the relative prognostic value of TNF-α and other clinical and pathological variables have not been determined. For patients with cancer, high serum TNF-α levels have recently been shown to correlate with the severity of malignant disease. 22 In addition, serum TNF-α levels have been reported to be of prognostic value in human colorectal cancer. 24 In this study, the mean serum TNF-α level in patients with untreated O-SCC was significantly higher than in the healthy controls. In addition, the positive rate for serum TNF-α among patients with O-SCC was significantly higher than that for healthy controls. These results suggest that the serum TNFα level is of diagnostic value in O-SCC. Patients with O-SCC were grouped according to T category, pathological N category, clinical stage, and pathological parameters to examine the relationship between serum TNF-α levels and each of these factors. There were no significant differences between the various T categories or stages. However, serum TNF-α levels were lower in patients who had metastatic lymph nodes (pN+) than in the patients without metastatic lymph nodes (pN-). Therefore, patients who present with a low level of serum TNF-α should probably undergo a careful examination for regional lymph node metastasis. Wada et al reported that an increase in the serum TNF-α level was thought to be involved in various inflammatory reactions and also played a role in the immune system.25 Recently, neutrophils have also been found to produce large quantities of cytokines 152
such as TNF-α, interleukin-1, and interleukin-8. It is postulated that these cytokines trigger each other in the cytokine network and thus exert a synergistic effect. In addition, some authors suggested that the serum TNF-α level may be affected by the activity of neutrophils and lymphocytes that are present locally and in peripheral blood.24-26 It is known that the tumour inhibitory activity of TNF-α decreases along with a decrease in neutrophils.22,24 Yamazaki and Okutomi stated that it may be possible that low-function neutrophils cannot produce TNF-α and thus work against the antitumour effects of TNF-α.26 The results of this histopathological study showed a significant correlation between serum TNFα levels in patients with O-SCC and the degree of keratinisation of the cancer cells, mode of invasion, and degree of histological malignancy. The serum TNF-α levels of patients with marked lymphoplastic infiltration were significantly higher than those of the slight and moderate lymphoplastic infiltration groups. These results suggest that the actions of TNF-α are also closely correlated with prognosis. When the relationship between the clinical course and the serum TNF-α level was examined among patients with O-SCC, the mean serum concentration of TNF-α was significantly higher among patients with a good clinical course than in the poor-clinical course group. Patients with a poor clinical course often had advanced O-SCC at the time of diagnosis. Moreover, TNF-α-negative patients with O-SCC were also found to have a significantly shorter overall survival than TNF-α-positive patients. These findings show that the serum TNF-α level is an independent prognostic marker for O-SCC. The use of CEA, SCCA, IAP, and a combination assay of these tumour markers in O-SCC are now accepted as a valuable tool for diagnosis, as well as determining prognosis and treatment monitoring.5-7,12 Although no significant difference was observed between the CEA-negative and the CEA-positive patients in relation to the serum TNF-α levels, the serum TNF-α levels in SCCA-negative patients were significantly lower than those in SCCA-positive Asian J Oral Maxillofac Surg Vol 14, No 3, 2002
Kurokawa, Takeda, Yamashita, et al
patients. On the other hand, the serum TNF-α levels in the IAP-negative patients were significantly higher than those in IAP-positive patients. These results suggest that, not only these tumour markers, but also serum TNF-α levels may be useful for screening patients with O-SCC. Moreover, the serum TNF-α levels appear to be affected by the ability of the tumour-bearing host to produce TNF-α and the activation of the immune system.
References 1. Socher SH, Martinez D, Craig JB, Kuhn JG, Ollif A. Tumor necrosis factor not detectable in patients with clinical cancer cachexia. J Natl Cancer Inst 1988;80:595-598. 2. Philips R, Epstein LB. Tumor necrosis factor as immunomodulator and mediator of monocyte cytotoxicity induced by itself, α-interferon and interleukin-4. Nature 1986;323:86-89. 3. Watanabe N, Umetsu T, Sone H, Neda H, Yamauchi N, Umeno H, Maeda M, Niitsu Y. Stimulation of antitumorigenic cytotoxicity in macrophages by tumor necrosis factor. Cancer J 1988;2:165-168. 4. Takeda K, Iwamoto S, Sugimoto H, Takuma T, Kawatani N, Noda M, Masaki A, Morise H, Arimura H, Konno K. Identity of differentiation inducing factors and tumor necrosis factors. Nature 1986;323:338-340. 5. Kurokawa H, Tsuru S, Okada M, Nakamura T, Kajiyama M. Evaluation of tumor markers in patients with squamous cell carcinoma in the oral cavity. Int J Oral Maxillofac Surg 1993;22:35-38. 6. Silverman NA, Alexander JC, Chretien PB. CEA levels in head and neck cancer. Cancer 1976;37: 2204-2209. 7. Fischbach N, Meyer T, Barthel K. Squamous cell carcinoma antigen in the diagnosis and treatment follow-up of oral and facial squamous cell carcinoma. Cancer 1990;65:1321-1326. 8. Kobayashi Y, Amagasa T, Fujii E, Yamashiro M. Evaluation of plasma Glutathione S-Transferaseπ (GST-π) as a tumor marker for oral squamous cell carcinoma. Int J Clin Oncol 1996;1:44-49. 9. Urada M, Komatsu M, Yamaoka M, Fukusawa K, Harada M, Mima T, Matsuya T. Serum dipeptidyl peptidase activities as a possible marker of Asian J Oral Maxillofac Surg Vol 14, No 3, 2002
oral cancer. Cancer 1989;64:1274-1277. 10. Doweck I, Barak M, Greenberg E. Cyfra 21-1. A new potential tumor marker for squamous cell carcinoma of head and neck. Arch Otolaryngol Head Neck Surg 1995;121:177-181. 11. Kurokawa H, Tokudome S, Yamashita Y, Ishibashi H, Yoshikawa T, Kajiyama M. Serum fragment of cytokeratin subunit 19 (cyfra) as a tumor marker in diagnosis and monitoring of treatment of oral squamous cell carcinoma. Int J Clin Oncol 1999;4:17-21. 12. Kurokawa H, YamashitaY, Tokudome S, Kajiyama M. Combination assay for tumor markers in oral squamous cell carcinoma. J Oral Maxillofac Surg 1997;55:964-966. 13. Hermanek P, Sobin LH. UICC International Union Against Cancer. TNM Classification and Grading of Malignant Tumors. Berlin: SpringerVerlag; 1987:27-39. 14. Thomson DMP, Krupey J, Freedman SO. The radioimmunoassay of circulating carcinoembryonic antigen of the human digestive system. Proc Natl Acad Sci USA 1969;64:16-19. 15. Meiyer W, Eiermann W, Stieber P. Experiences with SCC antigen, a new tumor marker for cervical carcinoma. Eur J Cancer Clin Oncol 1989; 25:235-238. 16. Mancini G, Carbonara AP, Hermans JE. Immunochemical quantitation of antigens by single radio immunodiffusion. Immunochemistry 1965;2:235-239. 17. Vincenz, K, Schoenthal E, Zekert F, Wunderer S. Diagnosis of head and neck carcinomas by means of immunological tumor markers. J Cranio Maxillofac Surg 1987;15:270-281. 18. Anneroth G, Batsakis J. review of the literature and a recommended system of malignancy grading in oral squamous cell carcinoma. Scand J Dent Res 1987:95:229-249. 19. Yamamoto E, Kohama G, Sunakawa H, Iwai M, Hiratsuka H. Mode of invasion, bleomaycin sensitivity, and clinical course in squamous cell carcinoma of the oral cavity. Cancer 1983;51: 2175-2182. 20. Trac KJ, Vlassara H, Cerami A. Cachectin/tumor necrosis factor. Lancet 1989;1:1122-1126. 21. Amman AJ, Palladino MA, Volberding P, Abrams D, Martin NL, Conant M. Tumor necrosis factors alpha and beta in acquired immunodeficiency 153
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syndrome (AIDS) and Aids-related complex. J Clin Immunol 1987;7:481-489. 22. Calandra T, Baumgartner JD, Grau GE, Wu MM, Lambert PH, Schellekens J, Verhoef J, Glayser M.P. Prognostic values of tumor necrosis factor/ cachectin, interleukin-1, interferon-α, and interferon-γ in the serum of patients with septic shock. J Infect Dis 1990;161:982-987. 23. Kurokawa H, Yamashita Y, Takeda S. Estimation of tumor necrosis factor alpha and manganese superoxide dismutase in oral squamous cell carcinoma — available methods for predicting the prognosis and monitoring the treatment. Asian J Oral Maxillofac Surg 2000;12:141-148.
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24. Satomi A, Murakami S, Hashimoto T, Okamura U, Okubo K, Takahashi S, Hirayama R, Sonoda M, Komada T. Estimation of serum tumor necrosis factor alpha and tumor tissue superoxide dismutase activity in the prognosis of human colorectal cancer. Int J Clin Oncol 1997;2:15-20. 25. Wada H, Minamikawa K, Kaneko T. Elevated plasma levels of cytokines in patients with idiopathic thrombocytopenic purpura. Mie Med J 1991;41:111-115. 26. Yamazaki M, Okutomi T. Augmentation of release of cytotoxin from murine bone marrow and peritoneal macrophages by tumor transplantation. Cancer Res 1989;49:352-356.
Asian J Oral Maxillofac Surg Vol 14, No 3, 2002
Asian J Oral Maxillofac Surg 2002;14:148-154.
Estimation of Serum Tumour Necrosis Factor-α and Correlation to Tumour Markers in Patients with Oral Squamous Cell Carcinoma Hideo Kurokawa,1 Shinobu Takeda,1 Yoshihiro Yamashita,1 Takashi Nakamura,1 Tomoyuki Murata,1 Tetsu Takahashi,1 Hiroshi Fukuyama,2 Min Zhang,2 Hiroaki Ishibashi3 1 Second Department of Oral and Maxillofacial Surgery, Kyushu Dental College, Kitakyushu, Japan 2 Department of Oral Pathology, Kyushu Dental College, Kitakyushu, Japan 3 Second Department of Oral and Maxillofacial Surgery, School of Dentistry, Kyushu University, Fukuoka, Japan
Abstract
Objectives: Estimation of serum tumour necrosis factor-α levels and correlation with tumour markers for the prognosis of oral squamous cell carcinoma before treatment. Patients and Methods: Serum levels of tumour necrosis factor-α and 3 other tumour markers — carcinoembryonic antigen, squamous cell carcinoma antigen, and immunosuppressive acidic protein — were simultaneously measured in 38 patients with oral squamous cell carcinoma and in 36 healthy controls. Results: The mean serum concentration of tumour necrosis factor-α for patients with oral squamous cell carcinoma (14.1 ± 13.2 pg/mL) was significantly higher than that of healthy controls (5.2 ± 3.1 pg/mL) [p < 0.05]. The mean serum concentration of tumour necrosis factor-α was lower for patients who were pathological node-positive than for pathological node-negative patients. A significant correlation was observed between the serum tumour necrosis factor-α levels and pathological parameters. The mean serum concentration of tumour necrosis factor-α was also significantly higher for patients who had a good clinical course (16.7 ± 18.6 pg/mL) than for those with a poor clinical course (9.6 ± 5.9 pg/mL) [p < 0.05]. The survival curve revealed a better prognosis for serum tumour necrosis factor-α-positive patients than for serum tumour necrosis factor-α -negative patients. A significant correlation was observed between the serum tumour necrosis factor-α levels and the tumour markers, squamous cell carcinoma antigen and immunosuppressive acidic protein (p < 0.05). Conclusion: An evaluation of serum tumour necrosis factor-α level in patients with oral squamous cell carcinoma appears to be a valuable screening tool for the prognosis and treatment of patients with this cancer. Key Words: Tumour necrosis factors, Carcinoma, squamous cell, Tumour markers, biological, Prognosis
Introduction Tumour necrosis factor-α (TNF-α), a product of activated macrophages, is an important mediator in cytoprotection.1 TNF-α has been reported to exert an anti-tumour effect through both an indirect mechanism and via the activation of such immunologically competent cells as neutrophils and macrophages.2,3 Correspondence: Hideo Kurokawa, DDS, PhD, Second Department of Oral and Maxillofacial Surgery, Kyushu Dental College, 2-6-1 Manazuru, Kokurakita-ku, Kitakyushu City, 803-8580, Japan. Tel: (81 93) 582 1131, Fax: (81 93) 582 1139 E-mail: [email protected]
148
Estimates of the serum TNF-a level in cancer patients prior to surgery vary greatly,4 but there have been few studies on the usefulness of such determinations in establishing the diagnosis and prognosis of oral squamous cell carcinoma (O-SCC). Other tumour markers that have been used for patients with O-SCC include carcinoembryonic antigen (CEA),5,6 squamous cell carcinoma antigen (SCCA),5,7 immunosuppressive acidic protein (IAP),5 glutathione S-transferase-π (GST-π),8 dipeptidylpeptidase (DPP),9 and fragment of cytokeratin subunit 19 (Cyfra).10,11 A combination assay of CEA, Asian J Oral Maxillofac Surg Vol 14, No 3, 2002
Kurokawa, Takeda, Yamashita, et al
SCCA, and IAP has been reported to be useful for screening patients with O-SCC. 12 This study evaluated the use of preoperative serum TNF-a levels as a means of establishing both the diagnosis and postoperative prognosis of O-SCC. In addition, the relationships between the serum TNF-a level and that of the tumour markers CEA, SCCA, and IAP was investigated.
Patients and Methods Thirty eight consecutive patients with O-SCC, who were treated at Kyushu Dental College in Japan between 1989 and 1992, were entered into the study. The patients ages ranged from 34 to 89 years (mean, 63.8 years) — 23 were men and 15 were women. The anatomic sites of O-SCC were the tongue (n = 15), mandibular gingiva (n = 11), maxillary gingiva (n = 9), floor of the mouth (n = 2), and buccal mucosa (n = 1). Preoperatively, all patients were categorised according to the International Union Against Cancer (UICC) TNM classification (1987),13 as follows: • 9 patients had T1 • 11 patients had T2 • 7 patients had T3 • 11 patients had T4 • 26 patients had N0 • 12 patients had N1 or N2 • all patients had M0. According to the stage of disease, 8 patients demonstrated stage I disease, 9 had stage II disease, 8 had stage III disease, and 13 had stage IV disease. All patients received combined preoperative chemotherapy with cis-platinum (160 mg), peplomycin (50 mg), and tegafur uracil (300 to 600 mg/ day). The patients were separated into 2 groups after treatment. Sixteen patients had a good clinical course and remained completely free of the disease for more than 5 years after treatment of the primary tumour. The poor-clinical course group consisted of 22 patients who had recurrent tumours at the primary site, or died due to local recurrence at the primary site and either regional lymph node metastasis or distant metastasis. The control group comprised 36 healthy individuals. The mean age was 61.3 years (range, 22 to 82 years) — 20 were male and 16 were female. Asian J Oral Maxillofac Surg Vol 14, No 3, 2002
Blood specimens were taken after informed consent was obtained. The sera were centrifuged for 20 minutes at 2000 rpm, and then stored at -80°C until analysis. The serum TNF-α levels were measured with an enzyme-linked immunosorbent assay (ELISA) using a highly sensitive TNF assay kit (Tore Fuji Bionics, Tokyo, Japan). One monoclonal antibody was immobilised in each well of a 96-well microplate. After the plates were washed with phosphate-buffered saline (PBS), 200 μL of standard and test samples were allowed to react with this antibody and were incubated for 2 hours at room temperature. After 3 washes, a second monoclonal antibody was added to each well and incubated for 2 hours at room temperature. The samples were washed again and 200 μL of substrate was then added and incubated for 30 minutes at room temperature in the dark. The absorbance of the product was measured at 490 nm. The detection threshold of the serum TNF-α level was 1.9 pg/mL. Based on the receiver operating characteristic (ROC) curves for the serum TNF-α levels, a threshold of 6.5 pg/ml was chosen as the upper limit of the normal (ULN) — values above this threshold were defined as TNF-αpositive. The serum levels of CEA were determined by radioimmunoassay (CEA Roche Z Gel Kit, Roche Tokyo, Japan) originally developed by Thomson et al.14 The ULN was set at 2.5 ng/mL based on the findings of previous reports.5,6 The sensitivity of the test was 0.5 ng/mL. For the measurement of serum SCCA, a doubleantibody radioimmunoassay kit was used (SCC RIA BEAD, Dainabot Co, Tokyo, Japan). The ULN was considered to be 2.0 ng/mL.15 The sensitivity of the test was 0.7 ng/mL. IAP was quantitatively assayed in the sera by single radial immunodiffusion, as described by Mancini et al,16 using commercially available agarose plates. These plates, which contain rabbit anti-IAP, were bought from Kayaku Antibiotics Research Co. (Tokyo, Japan). The ULN was considered to be 500 μg/mL.17 149
Tumour Necrosis Factor-α in Oral Squamous Cell Carcinoma
Data were summarised as the mean activity plus or minus the standard deviation (A ± SD). A statistical analysis was performed using the arithmetic mean, the Mann-Whitney U test, and the chi-squared test. The survival rates were calculated by the KaplanMeier method, while the statistical difference was calculated by the log-rank test. A value of p < 0.05 was considered to be statistically significant.
Results Comparison of Serum Tumour Necrosis Factor- α Levels Between Patients and Controls The mean serum concentration of TNF-α was significantly higher in untreated patients with O-SCC than in healthy controls (p < 0.05). Twenty five of 38 patients (65.8%) were found to have elevated serum levels (Figure 1). Relationship Between Serum Tumour Necrosis Factor-α and Clinical Parameters The mean serum TNF-α level was 14.9 ± 10.3 pg/mL for T1/T2 (n = 20) and 10.5 ± 5.9 pg/mL for T3/T4 150
(pg/ml) 100 p < 0.05
TNF-α level
A histological examination was performed on the hematoxylin and eosin-stained sections prepared from either specimens biopsied before treatment or from surgical specimens. The histological malignancy grading system was determined according to Anneroth and Batsakis18 after evaluating the degree of keratinisation, nuclear polymorphism, number of mitosis, pattern of invasion, stage of invasion, and lymphoplasmatic infiltration. Each of these features was graded from 1 to 4 according to the definitions, giving a total malignancy score ranging from 6 to 24. The tumours were divided into 3 groups. The lowgrade malignancy group had a total malignancy score of <12, the moderate-grade malignancy group score ranged from 13 to 16, and the high-grade malignancy group had ≥17 points. All grading was performed independently by 2 pathologists. The relationships between the serum TNF-α levels, clinical (including T [T1/T2 and T3/T4] and pathological node [pN+ and pN-] categories) and stage classification (stages 1/2 and stages 3/4), and pathological parameters (including degree of keratinisation, lymphoplasmatic infiltration, mode of invasion,19 and histological malignancy) were evaluated.
Positive Negative
50
14.1±13.2 6.5
Positive rate
5.2±3.1
Healthy controls (n = 36) 11.1% (4 patients)
O-SCC patients (n = 38) 65.8% (25 patients)
p < 0.05
Figure 1. Comparison of serum tumour necrosis factor-α (TNF-α) levels among patients with oral squamous cell carcinoma (O-SCC) and healthy controls.
(n = 18) — the difference was not significant (Table 1). However, serum TNF-α levels were significantly lower in the patients who had metastatic lymph nodes (10.9 ± 9.8 pg/mL; n = 14) than in patients without metastatic lymph nodes (16.1 ± 16.2 pg/mL; n = 24) [p < 0.05; Table 1]. When the TNF-α level was analysed according to the UICC staging system,13 no significant differences were observed between the various stages (Table 1). Relationship Between Tumour Necrosis Factor- α and Pathological Parameters The mean serum TNF-α levels relative to the degree of keratinisation was 24.7 ± 38.4 pg/mL for the patients with a high degree of keratinisation (n = 24) Clinical parameters
Tumour necrosis factor-α level (pg/mL)
p Value
Tumour T1/T2 (n = 20) T3/T4 (n = 18)
14.9 ± 10.3 10.5 ± 5.9
NS
Node pN- (n = 24) pN+ (n = 14)
16.1 ± 16.2 10.9 ± 9.8
p < 0.05
Stage I/II (n = 17) III/IV (n = 21)
14.6 ± 9.5 13.1 ± 10.1
NS
Table 1. Relationship between serum tumour necrosis factor-α levels and the clinical parameters.
Asian J Oral Maxillofac Surg Vol 14, No 3, 2002
Kurokawa, Takeda, Yamashita, et al Tumour necrosis factor-α level (pg/mL)
p Value
Degree of keratinisation High (n = 24) Moderate (n = 14)
24.7 ± 38.4 8.5 ± 5.1
< 0.05
Lymphoplasmatic infiltration Slight, moderate (n = 24) Marked (n = 14)
10.5 ± 7.9 16.4 ± 19.4
< 0.05
Mode of invasion Grade 1 to 3 (n = 27) Grade 4C to 4D (n = 11)
18.4 ± 10.3 9.2 ± 6.6
< 0.05
Histological malignancy Low grade (n = 15) Moderate, high (n = 23)
20.1 ± 18.2 9.1 ± 6.4
< 0.05
Table 2. Relationship between serum tumour necrosis factor-α levels and pathological parameters.
and 8.5 ± 5.1 pg/mL for patients with a moderate degree of keratinisation (n = 14) — a significant difference between the 2 groups (p < 0.05). The mean serum TNF-α level relative to the degree of lymphoplasmatic infiltration was 10.5 ± 7.9 pg/mL for the slightly and moderately infiltrated patients (n = 24) and 16.4 ± 19.4 pg/mL for the markedly infiltrated group (n = 14), with a significant difference between them (p < 0.05). Regarding the mode of invasion, the mean serum TNF-α levels of the grades 1 to 3 groups (n = 27) were higher than for those with grades 4C (cord like pattern)/4D (diffuse pattern) [n = 11]. In addition, the mean serum TNF-α levels in patients with low histological malignancy were higher than those of the moderate and high histological malignancy group (Table 2). Relationship Between Tumour Necrosis Factor- α Levels and 5-Year Survival The mean serum concentration of TNF-α was significantly higher in patients with a good clinical course (16.7 ± 18.6 pg/mL, n = 16) than in patients with a poor clinical course (9.6 ± 5.9 pg/mL, n = 22) [p < 0.05]. The 5-year cumulative survival rate of the TNF-αpositive group was higher (80.4%) than that of the TNF-α-negative group (38.5%) [p < 0.05; Figure 2]. Relationship Between Tumour Necrosis Factor- α and Tumour Markers The relationship between serum TNF-α level and the other tumour markers is shown in Table 3. Although Asian J Oral Maxillofac Surg Vol 14, No 3, 2002
100
TNF-αpositive 80.4%
80
Survival rate (%)
Pathological parameters
60 TNF-αnegative 38.5%
40
20 p < 0.05 0
10
20
30
40
50
60
Time (months)
Figure 2. Overall actuarial survival curves by tumour necrosis factor-α (TNF-α).
no significant difference was seen between the CEA-negative group and the CEA-positive group with respect to the serum TNF-α level, the serum TNF-α level in the SCCA-negative group was significantly lower than in the SCCA-positive group (p < 0.05). The serum TNF-α level in the IAPnegative group was also significantly higher than in the IAP-positive group (p < 0.05).
Discussion TNF-α is a polypeptide cytokine produced by stimulated macrophages and lymphocytes.1 TNF-α was initially thought to be useful as an anticancer drug, free from side effects, but it was later shown to be identical to cachectin, and is now known to have a variety of biological activities.20 Many authors have Tumour marker
Tumour necrosis p Value factor-α level (pg/mL)
Carcinoembryonic antigen Positive (n = 16) Negative (n = 17)
10.1 ±6.1 14.4 ± 17.3
NS
Squamous cell carcinoma antigen Positive (n = 12) Negative (n = 21)
7.8 ± 5.5 15.0 ±15.4
< 0.05
Immunosuppressive acidic protein Positive (n = 12) 17.4 ± 18.5 Negative (n = 21) 8.9 ± 6.0
< 0.05
Table 3. Relationship between serum tumour necrosis factor-α levels and other tumour markers (carcinoembryonic antigen, squamous cell carcinoma antigen, and immunosuppressive acidic protein).
151
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reported that TNF-α acts directly against cancer cells by exerting antitumour effects through an indirect mechanism and via the activation of immunologically competent cells such as neutrophils and macrophages.2,3 A deficiency in TNF-α production by monocytes may therefore result in a susceptibility to infection, malignancy, and other physiological abnormalities.21 However, the mechanism of action of TNF-α in humans remains controversial. Many investigators have also found that the level of biologically active or immunoreactive TNF-α is associated with outcome in patients with malignant tumours,22,23 although the relative prognostic value of TNF-α and other clinical and pathological variables have not been determined. For patients with cancer, high serum TNF-α levels have recently been shown to correlate with the severity of malignant disease. 22 In addition, serum TNF-α levels have been reported to be of prognostic value in human colorectal cancer. 24 In this study, the mean serum TNF-α level in patients with untreated O-SCC was significantly higher than in the healthy controls. In addition, the positive rate for serum TNF-α among patients with O-SCC was significantly higher than that for healthy controls. These results suggest that the serum TNFα level is of diagnostic value in O-SCC. Patients with O-SCC were grouped according to T category, pathological N category, clinical stage, and pathological parameters to examine the relationship between serum TNF-α levels and each of these factors. There were no significant differences between the various T categories or stages. However, serum TNF-α levels were lower in patients who had metastatic lymph nodes (pN+) than in the patients without metastatic lymph nodes (pN-). Therefore, patients who present with a low level of serum TNF-α should probably undergo a careful examination for regional lymph node metastasis. Wada et al reported that an increase in the serum TNF-α level was thought to be involved in various inflammatory reactions and also played a role in the immune system.25 Recently, neutrophils have also been found to produce large quantities of cytokines 152
such as TNF-α, interleukin-1, and interleukin-8. It is postulated that these cytokines trigger each other in the cytokine network and thus exert a synergistic effect. In addition, some authors suggested that the serum TNF-α level may be affected by the activity of neutrophils and lymphocytes that are present locally and in peripheral blood.24-26 It is known that the tumour inhibitory activity of TNF-α decreases along with a decrease in neutrophils.22,24 Yamazaki and Okutomi stated that it may be possible that low-function neutrophils cannot produce TNF-α and thus work against the antitumour effects of TNF-α.26 The results of this histopathological study showed a significant correlation between serum TNFα levels in patients with O-SCC and the degree of keratinisation of the cancer cells, mode of invasion, and degree of histological malignancy. The serum TNF-α levels of patients with marked lymphoplastic infiltration were significantly higher than those of the slight and moderate lymphoplastic infiltration groups. These results suggest that the actions of TNF-α are also closely correlated with prognosis. When the relationship between the clinical course and the serum TNF-α level was examined among patients with O-SCC, the mean serum concentration of TNF-α was significantly higher among patients with a good clinical course than in the poor-clinical course group. Patients with a poor clinical course often had advanced O-SCC at the time of diagnosis. Moreover, TNF-α-negative patients with O-SCC were also found to have a significantly shorter overall survival than TNF-α-positive patients. These findings show that the serum TNF-α level is an independent prognostic marker for O-SCC. The use of CEA, SCCA, IAP, and a combination assay of these tumour markers in O-SCC are now accepted as a valuable tool for diagnosis, as well as determining prognosis and treatment monitoring.5-7,12 Although no significant difference was observed between the CEA-negative and the CEA-positive patients in relation to the serum TNF-α levels, the serum TNF-α levels in SCCA-negative patients were significantly lower than those in SCCA-positive Asian J Oral Maxillofac Surg Vol 14, No 3, 2002
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patients. On the other hand, the serum TNF-α levels in the IAP-negative patients were significantly higher than those in IAP-positive patients. These results suggest that, not only these tumour markers, but also serum TNF-α levels may be useful for screening patients with O-SCC. Moreover, the serum TNF-α levels appear to be affected by the ability of the tumour-bearing host to produce TNF-α and the activation of the immune system.
References 1. Socher SH, Martinez D, Craig JB, Kuhn JG, Ollif A. Tumor necrosis factor not detectable in patients with clinical cancer cachexia. J Natl Cancer Inst 1988;80:595-598. 2. Philips R, Epstein LB. Tumor necrosis factor as immunomodulator and mediator of monocyte cytotoxicity induced by itself, α-interferon and interleukin-4. Nature 1986;323:86-89. 3. Watanabe N, Umetsu T, Sone H, Neda H, Yamauchi N, Umeno H, Maeda M, Niitsu Y. Stimulation of antitumorigenic cytotoxicity in macrophages by tumor necrosis factor. Cancer J 1988;2:165-168. 4. Takeda K, Iwamoto S, Sugimoto H, Takuma T, Kawatani N, Noda M, Masaki A, Morise H, Arimura H, Konno K. Identity of differentiation inducing factors and tumor necrosis factors. Nature 1986;323:338-340. 5. Kurokawa H, Tsuru S, Okada M, Nakamura T, Kajiyama M. Evaluation of tumor markers in patients with squamous cell carcinoma in the oral cavity. Int J Oral Maxillofac Surg 1993;22:35-38. 6. Silverman NA, Alexander JC, Chretien PB. CEA levels in head and neck cancer. Cancer 1976;37: 2204-2209. 7. Fischbach N, Meyer T, Barthel K. Squamous cell carcinoma antigen in the diagnosis and treatment follow-up of oral and facial squamous cell carcinoma. Cancer 1990;65:1321-1326. 8. Kobayashi Y, Amagasa T, Fujii E, Yamashiro M. Evaluation of plasma Glutathione S-Transferaseπ (GST-π) as a tumor marker for oral squamous cell carcinoma. Int J Clin Oncol 1996;1:44-49. 9. Urada M, Komatsu M, Yamaoka M, Fukusawa K, Harada M, Mima T, Matsuya T. Serum dipeptidyl peptidase activities as a possible marker of Asian J Oral Maxillofac Surg Vol 14, No 3, 2002
oral cancer. Cancer 1989;64:1274-1277. 10. Doweck I, Barak M, Greenberg E. Cyfra 21-1. A new potential tumor marker for squamous cell carcinoma of head and neck. Arch Otolaryngol Head Neck Surg 1995;121:177-181. 11. Kurokawa H, Tokudome S, Yamashita Y, Ishibashi H, Yoshikawa T, Kajiyama M. Serum fragment of cytokeratin subunit 19 (cyfra) as a tumor marker in diagnosis and monitoring of treatment of oral squamous cell carcinoma. Int J Clin Oncol 1999;4:17-21. 12. Kurokawa H, YamashitaY, Tokudome S, Kajiyama M. Combination assay for tumor markers in oral squamous cell carcinoma. J Oral Maxillofac Surg 1997;55:964-966. 13. Hermanek P, Sobin LH. UICC International Union Against Cancer. TNM Classification and Grading of Malignant Tumors. Berlin: SpringerVerlag; 1987:27-39. 14. Thomson DMP, Krupey J, Freedman SO. The radioimmunoassay of circulating carcinoembryonic antigen of the human digestive system. Proc Natl Acad Sci USA 1969;64:16-19. 15. Meiyer W, Eiermann W, Stieber P. Experiences with SCC antigen, a new tumor marker for cervical carcinoma. Eur J Cancer Clin Oncol 1989; 25:235-238. 16. Mancini G, Carbonara AP, Hermans JE. Immunochemical quantitation of antigens by single radio immunodiffusion. Immunochemistry 1965;2:235-239. 17. Vincenz, K, Schoenthal E, Zekert F, Wunderer S. Diagnosis of head and neck carcinomas by means of immunological tumor markers. J Cranio Maxillofac Surg 1987;15:270-281. 18. Anneroth G, Batsakis J. review of the literature and a recommended system of malignancy grading in oral squamous cell carcinoma. Scand J Dent Res 1987:95:229-249. 19. Yamamoto E, Kohama G, Sunakawa H, Iwai M, Hiratsuka H. Mode of invasion, bleomaycin sensitivity, and clinical course in squamous cell carcinoma of the oral cavity. Cancer 1983;51: 2175-2182. 20. Trac KJ, Vlassara H, Cerami A. Cachectin/tumor necrosis factor. Lancet 1989;1:1122-1126. 21. Amman AJ, Palladino MA, Volberding P, Abrams D, Martin NL, Conant M. Tumor necrosis factors alpha and beta in acquired immunodeficiency 153
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syndrome (AIDS) and Aids-related complex. J Clin Immunol 1987;7:481-489. 22. Calandra T, Baumgartner JD, Grau GE, Wu MM, Lambert PH, Schellekens J, Verhoef J, Glayser M.P. Prognostic values of tumor necrosis factor/ cachectin, interleukin-1, interferon-α, and interferon-γ in the serum of patients with septic shock. J Infect Dis 1990;161:982-987. 23. Kurokawa H, Yamashita Y, Takeda S. Estimation of tumor necrosis factor alpha and manganese superoxide dismutase in oral squamous cell carcinoma — available methods for predicting the prognosis and monitoring the treatment. Asian J Oral Maxillofac Surg 2000;12:141-148.
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24. Satomi A, Murakami S, Hashimoto T, Okamura U, Okubo K, Takahashi S, Hirayama R, Sonoda M, Komada T. Estimation of serum tumor necrosis factor alpha and tumor tissue superoxide dismutase activity in the prognosis of human colorectal cancer. Int J Clin Oncol 1997;2:15-20. 25. Wada H, Minamikawa K, Kaneko T. Elevated plasma levels of cytokines in patients with idiopathic thrombocytopenic purpura. Mie Med J 1991;41:111-115. 26. Yamazaki M, Okutomi T. Augmentation of release of cytotoxin from murine bone marrow and peritoneal macrophages by tumor transplantation. Cancer Res 1989;49:352-356.
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