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Prognostic Significance of Vascular Endothelial Growth Factor Protein Levels in Oral and Oropharyngeal Squamous Cell Carcinoma

From the Departments of Therapeutic Radiology, Pathology, and Otolaryngology, Yale School of Medicine, New Haven, CT.

Address reprint requests to Bruce G. Haffty, MD, Department of Therapeutic Radiology, Yale University School of Medicine, New Haven, CT 06520-8040; email bruce.haffty{at}yale.edu

	ABSTRACT

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PURPOSE: Vascular Endothelial Growth Factor (VEGF) promotes angiogenesis in many different tumor types. VEGF levels may affect tumor growth, metastatic potential, and response to radiotherapy. This study assesses the prognostic value of VEGF protein levels in a cohort of patients with oral and oropharyngeal squamous cell carcinomas. The relationships between clinical outcome and the covariables of tumor-node-metastasis stage, disease stage (I to IV), grade, margin status, race, sex, and age were also determined.

PATIENTS AND METHODS: Chart review identified 77 patients with oral or oropharyngeal squamous cell carcinoma treated with gross total surgical resection and postoperative radiation between 1981 and 1992. Sufficient follow-up data and tumor tissue were available in 56 patients (73%). VEGF protein levels were determined using immunohistochemistry. The association between VEGF status, covariables, and outcome was assessed in a bivariate and multivariate model using two-sided statistical tests.

RESULTS: Twenty-three tumors (41%) were positive for VEGF expression. VEGF-positive tumors were more likely to recur locally (relative risk [RR] = 3.08; 95% confidence interval [CI], 1.03 to 9.24) and distantly (RR = 4.62; 95% CI, 1.41 to 15.10). In bivariate analysis, VEGF positivity was the most significant predictor of poor disease-free survival (RR = 2.66; 95% CI, 1.27 to 5.56) and overall survival (RR = 3.21; 95% CI, 1.63 to 6.32). In multivariate analysis, VEGF positivity was the most significant predictor of poor disease-free survival (RR = 2.75; 95% CI, 1.30 to 5.79) and overall survival (RR = 3.53; 95% CI, 1.75 to 7.13).

CONCLUSION: In this cohort, VEGF positivity was the most significant predictor of poor prognosis. VEGF status may prove to be an important prognostic factor in head and neck cancer.

	INTRODUCTION

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EPITHELIAL NEOPLASMS derived from the mucosa of the oral cavity and oropharynx account for 30,000 new cases of cancer each year in the United States.¹ These tumors form a subset within the broader category of head and neck squamous cell carcinoma that includes tumors derived from the oral cavity, oropharynx, hypopharynx, nasopharynx, and larynx. Despite extensive research into the pathogenesis and management of oral and oropharyngeal squamous cell carcinomas, the 5-year survival rate for patients with these tumors has not improved in the last 25 years, remaining at 53%.¹ Currently, treatment decisions in oral and oropharyngeal tumors are guided by clinical-pathologic factors such as age, sex, race, tumor-node-metastasis stage, and histologic grade. Although useful, these factors fail to provide definitive information regarding the overall aggressiveness of a tumor and its potential to recur. In theory, a factor that consistently identified patients at risk for recurrent disease would help to improve disease-free survival by allowing physicians to select high-risk patients for more aggressive treatment. In addition, identification of a biologic marker of aggressive disease would help to provide new avenues for rational drug design targeted against specific molecular defects.

In recent years, a number of biologic markers have been identified that may provide prognostically useful information in the management of head and neck squamous cell carcinoma.² Mutation of the tumor suppressor gene p53,^3-6 amplification of the proto-oncogene cyclin D1,^7-13 and overexpression of the tyrosine kinase receptor for epidermal growth factor^14-17 have all been associated with poor prognosis.

Markers related to tumor neovascularization may also predict outcome in head and neck cancer patients. To grow beyond microscopic size, both primary tumors and their micrometastases must initiate angiogenesis.^18,19 Vascular endothelial growth factor (VEGF) promotes tumor angiogenesis in head and neck cancer and many other tumor types by stimulating endothelial-cell proliferation and promoting vascular permeability.^19-21 VEGF is a 34- to 50-kd dimer composed of two identical disulfide-linked subunits that arise from differential splicing of a single gene.²² It is induced under hypoxic conditions in a wide variety of tissue types and binds to the receptor tyrosine kinases Flt-1 and KDR/Flk-1.^23-27 Because hypoxic tumors display increased radioresistance, VEGF protein levels may serve as a surrogate marker for hypoxic radioresistance.

Support for the relationship between tumor angiogenesis and response to treatment has derived from two recent studies in node-negative breast carcinoma that reported a significant correlation between VEGF protein levels and poor prognosis in a multivariate model.^28,29 Currently, the literature linking VEGF expression to prognosis in head and neck cancer is less complete. Two studies conducted on cohorts of 45 and 36 patients reported an association between VEGF protein levels and prognosis in a bivariate model,^30,31 but one study with a cohort of 156 found no relationship between VEGF levels and overall survival.³²

The aim of this study was to determine the prognostic significance of VEGF protein levels in squamous cell carcinoma of the head and neck. In an effort to ensure homogeneity and enhance the validity of the study, the patient population selected was limited to patients with advanced but resectable squamous cell carcinoma of the oral cavity and oropharynx treated with gross total surgical resection and postoperative external-beam radiation therapy. Tumors from other sites of the head and neck and tumors managed with other therapeutic strategies were excluded from the study. In addition, we sought to compare the prognostic utility of VEGF protein levels with traditional prognostic markers, including tumor-node-metastasis stage, overall stage, tumor grade, margin status, race, sex, and age at diagnosis.

	PATIENTS AND METHODS

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Patients
Criteria for patient inclusion in this study were as follows: (1) presentation to the Department of Therapeutic Radiology at Yale School of Medicine between 1981 and 1992 with a histologically confirmed diagnosis of squamous cell carcinoma in the oral cavity or oropharynx, and (2) treatment with primary surgical excision and postoperative external-beam radiotherapy with curative intent. Patients were excluded from this study if they had a prior history of head and neck squamous cell carcinoma or if they failed to receive a full course of radiation therapy. A review of radiation records identified 77 patients who met the entry criteria. Of these, 14 patients had incomplete follow-up, and 10 tissue samples were unavailable, leaving 56 patients for inclusion in the study.

With the approval of the Yale School of Medicine Human Investigations Committee and the West Haven Veterans Affairs Hospital Human Studies Committee, paraffin-embedded tissue samples were obtained from the appropriate pathology department archives. Covariables, including demographics, staging, and clinical, pathologic, and treatment parameters, were extracted from patient charts. Follow-up information was tabulated with respect to local, regional, and distant recurrence, the occurrence of a second primary tumor, and patient survival. Forty-one patients (73%) were observed until death, and the median follow-up time of the surviving patients was 6.1 years, with a minimum of 2.8 years.

All patients were treated with gross total surgical resection and postoperative external-beam radiotherapy to a median dose of 60 Gy. Final surgical margins were negative in 42 patients (75%). Forty-nine patients (88%) received a radical or modified neck dissection. Eleven patients (20%) received adjuvant chemotherapy with the hypoxic cytotoxins mitomycin or porfiromycin as part of an experimental protocol.^33,34 Five patients (9%) received intraoperative brachytherapy. All patients were staged clinically according to the American Joint Committee on Cancer tumor-node-metastasis staging system.³⁵ Clinically N0 patients were restaged for the purposes of this analysis if pathologic examination of the neck dissection specimen was positive for nodal metastases.

Immunohistochemical Analysis of Tissue Sections for the Presence of VEGF
Immunohistochemical analysis was performed on 5-µm–thick tissue sections prepared from formalin-fixed, paraffin-embedded archival tissue from the resected primary tumor. Tissue sections were deparaffinized in xylene followed by 100% ethanol. Samples were then quenched in 2% hydrogen peroxide/methanol solution followed by gradual hydration. Samples were pretreated to promote antigen retrieval by microwaving at 50% power three times for 5 minutes in 10 mmol/L of pH 6.0 sodium citrate. After antigen retrieval, slides were incubated in 1% bovine serum albumin diluted in phosphate buffered saline (PBS) for 5 minutes followed by a 30-minute incubation in suppressor serum (1:200 dilution normal horse serum in PBS). Slides were incubated overnight at 4°C with a mouse monoclonal Immunoglobulin G₁ reactive with the 34- to 50-kd isoforms of VEGF (1:200 dilution in PBS; Oncogene Research Products, Boston, MA; clone JH121). The next day, slides were washed in PBS and 0.01% Triton/PBS and then incubated for 30 minutes in horse antimouse secondary antibody (1:200 in PBS). After washes in PBS and 0.01% Triton/PBS, samples were labeled with ABC Elite streptavidin-peroxidase (Vector Laboratories, Inc, Burlingame, CA). After washing in PBS and 0.01% Triton/PBS, slides were incubated with the chromogen diaminobenzidine tetrahydrochloride (filtered before use), counterstained with hematoxylin, and mounted.

An experienced pathologist (D.C.), blinded to the clinical outcomes, examined multiple microscopic fields to score the tissue sections for tumor VEGF staining intensity (0 = none, 1 = faint and focal, 2 = moderate, 3 = strong, and 4 = intense) and percent distribution. For the analysis presented in this article, tumor VEGF positivity was defined as strong or intense immunohistochemical staining. As a negative control, the primary antibody incubation step was omitted in the staining of a tumor section known to be VEGF positive.

Statistical Analysis
VEGF status and relevant covariables were assembled in a database and analyzed using SAS User’s Guide, Version 6.12 (SAS Institute, Cary, NC). All tests of statistical significance were two-sided. Follow-up time and time to recurrence were calculated from the date of surgery to the date of the relevant outcome. Disease-free survival was calculated as the interval between the date of surgery and the date of the first recurrence of disease.

Bivariate analyses for the association between covariables and VEGF positivity included the ² test and the Fisher’s exact test. Bivariate analyses for the associations between predictor variables and local, regional, and distant recurrence, disease-free survival, and overall survival were conducted using the log-rank test and the ² test for linear trend. Variables violating the linearity assumption entered analysis as a categorical variable, with cut points determined by quartiles. Unadjusted relative risks (RRs) were calculated by entering variables into the Cox proportional hazards model.

In the multivariate analysis, Cox proportional hazards regression determined significant predictors of disease-free survival and overall survival at an = 0.05 level in the final model. The predictor and all covariables were initially entered into a forward parsimonious selection. Variables with P .1 were retained for the final model.

	RESULTS

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Descriptive Statistics
A total of 77 patients were eligible for the analysis, but 21 were excluded because of incomplete follow-up data, leaving 56 patients (73%) included in the analysis. Frequency statistics for clinical-pathologic characteristics of the patient cohort are listed in Table 1.

View larger version (133K): [in this window] [in a new window]   Fig 1. VEGF immunohistochemical staining of formalin-fixed, paraffin-embedded tumor tissue. (A) 0+ (no) staining; (B) 2+ (moderate) staining; (C) 3+ (strong) staining; (D) negative control, tumor from sample C stained without primary antibody directed to VEGF (400x magnification, scale bar = 50 µm).

Bivariate Analysis
The results of the Fisher’s exact tests and ² tests for associations between VEGF positivity and covariables are listed in Table 1. None of the variables were significantly associated with VEGF positivity at = 0.05. Results from the log-rank test for associations between predictor variables and disease-free survival and overall survival are listed in Table 1. The main predictor, VEGF positivity, was significantly associated with any recurrence (P = .007) and overall survival (P = .0004). Unadjusted RRs were calculated for bivariate associations. For any recurrence, the unadjusted RR for VEGF-positive status was 2.66 (95% confidence interval [CI], 1.27 to 5.56). For death, the unadjusted RR was 3.21 (95% CI, 1.63 to 6.32). Kaplan-Meier survival curves for disease-free and overall survival with respect to VEGF status are presented in Fig 2.

View larger version (17K): [in this window] [in a new window]   Fig 2. Kaplan-Meier survival curves for disease-free and overall survival with respect to VEGF status.

Log-rank statistics for local recurrence indicated a significant association with VEGF positivity, with 16% of VEGF-negative tumors (six of 33 tumors) recurring locally versus 35% of VEGF-positive tumors (eight of 23 tumors) recurring locally (RR = 3.08; 95% CI, 1.03 to 9.24; P = .04). VEGF-positive tumors were also more likely to recur distantly, with 12% of VEGF-negative tumors (four of 33 tumors) recurring distantly versus 43% of VEGF-positive tumors (10 of 23 tumors) recurring distantly (RR = 4.62; 95% CI, 1.41 to 15.10; P = .006). VEGF status did not correlate with regional recurrence.

Multivariate Analysis
Both Cox proportional hazards models included all 56 patients. VEGF positivity and the aforementioned covariables were entered into the model. For disease-free survival, the variables of race and VEGF positivity were retained at a level of 0.10 (Table 2). With a final = 0.05 criterion for significance, only VEGF positivity significantly predicted disease-free survival (RR = 2.75; 95% CI, 1.30 to 5.79; P = .0078). Thus, patients with VEGF-positive status were almost three times as likely to experience recurrence compared with patients with VEGF-negative status.

	DISCUSSION

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

 
In this study, a uniform cohort of patients with squamous cell carcinoma of the oral cavity and oropharynx treated with gross total surgical resection and postoperative external-beam radiotherapy was selected to assess the prognostic value of VEGF immunohistochemical staining. VEGF-positive tumors were more likely to recur both locally and distantly. In addition, VEGF positivity was strongly correlated with poor disease-free and overall survival. In multivariate analysis, VEGF positivity was the most significant predictor of poor disease-free and overall survival. Thus, in comparison with standard clinical-pathologic predictors of outcome, such as tumor-node-metastasis stage, overall stage, tumor grade, margin status, sex, race, and age, VEGF status was a better predictor of outcome in this cohort.

In addition to VEGF status, race was also a significant predictor of overall survival in multivariate analysis. The deleterious effect of black race on overall survival is consistent with other studies in head and neck cancer showing black race to be a risk factor for poor survival³⁶ and for potentially fatal surgical wound infection.^37,38

This study was limited to a homogeneous cohort to control for variations in tumor behavior imposed by different primary tumor sites and treatment modalities. These criteria enhanced study validity while limiting sample size and power. As a result, larger studies will be needed to confirm these initial findings and further elucidate the relative importance of VEGF status in comparison with tumor stage.

The positive correlation between VEGF protein levels and prognosis reported in this study echoes the results obtained in two recent studies of angiogenesis promoters in breast cancer. In one study conducted on 305 primary breast tumors, VEGF protein levels were an independent predictor of relapse-free survival.²⁹ In another study of 525 node-negative breast carcinoma patients (T1-2N0M0), VEGF levels were the strongest predictor of overall survival in a multivariate model.²⁸

To date, the literature on angiogenesis in head and neck cancer is more limited and less conclusive. A study of VEGF levels in 45 oral squamous cell carcinoma patients treated with radiotherapy alone or preoperative radiotherapy plus surgical resection revealed a significant relationship between VEGF positivity and poor overall survival in bivariate analysis.³¹ In an additional study conducted on 36 patients undergoing primary surgical resection for head and neck squamous cell carcinoma, high VEGF levels predicted a higher rate of disease recurrence and shorter disease-free interval in bivariate analysis.³⁰ In contrast, a study of 156 patients with head and neck squamous cell carcinoma treated with surgical excision and postoperative radiotherapy failed to find a significant relationship between VEGF positivity and overall survival and did not report the relationship between VEGF positivity and disease-free survival.³² The variability in these findings may be explained by a number of factors, including differences in staining methods, broad inclusion criteria for tumor sites, patient populations, and therapeutic strategies. Taken as a whole, however, the majority of the literature from breast and head and neck cancer supports the hypothesis that high VEGF levels predict aggressive disease.

Angiogenesis may promote metastatic disease by exposing tumors to a greater endothelial surface area, thus increasing the likelihood of hematogenous dissemination.³⁹ In addition, once a tumor has formed a distant micrometastasis, it must recruit a vascular supply to proliferate to a clinically significant size. These hypotheses are consistent with the strong association in this study between VEGF protein levels and distant recurrence.

In addition to promoting metastatic disease, VEGF protein levels may identify tumors with increased resistance to radiotherapy. Because VEGF is induced under hypoxic conditions, and hypoxic tumors tend to display increased radioresistance, VEGF protein expression may serve as a surrogate marker of hypoxic radioresistance. This hypothesis is consistent with the correlation between VEGF positivity and local recurrence observed in this study. Future studies assessing the prognostic value of VEGF in cohorts treated solely with radiotherapy will be necessary to confirm or deny the putative relationship between VEGF expression and tumor radioresistance.

The results of this study, coupled with the existing literature, suggest that tumors with high VEGF protein levels may be at greater risk for poor disease-free survival, poor overall survival, and metastatic disease. If these results are confirmed in additional studies, VEGF status could emerge as an important factor in establishing prognosis and selecting treatment modalities for oral cavity and oropharyngeal squamous cell carcinomas. In addition, therapies targeted at the molecular mechanism of angiogenesis, such as ligands that antagonize the interaction of VEGF with its receptors or antibodies directed against VEGF and its receptors, may prove useful in the management of oral cavity and oropharyngeal cancers. Studies such as this that evaluate the relationship between specific molecular processes and clinical outcome will prove essential in the identification of pathways that will serve as optimal targets for the next generation of cancer pharmacotherapies.

	NOTES

 
Yale University School of Medicine Medical Student Research Training Fellowship, Yale Comprehensive Cancer Center Clinical Studies Support Award.

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Submitted October 12, 1999; accepted January 26, 2000.

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				E. P. Hui, A. T. C. Chan, F. Pezzella, H. Turley, K.-F. To, T. C. W. Poon, B. Zee, F. Mo, P. M. L. Teo, D. P. Huang, et al. Coexpression of Hypoxia-inducible Factors 1{alpha} and 2{alpha}, Carbonic Anhydrase IX, and Vascular Endothelial Growth Factor in Nasopharyngeal Carcinoma and Relationship to Survival Clin. Cancer Res., August 1, 2002; 8(8): 2595 - 2604. [Abstract] [Full Text] [PDF]

				A. J. Khan, B. L. King, B. D. Smith, G. L. Smith, M. P. DiGiovanna, D. Carter, and B. G. Haffty Characterization of the HER-2/neu Oncogene by Immunohistochemical and Fluorescence in Situ Hybridization Analysis in Oral and Oropharyngeal Squamous Cell Carcinoma Clin. Cancer Res., February 1, 2002; 8(2): 540 - 548. [Abstract] [Full Text] [PDF]

				R. Masood, J. Cai, T. Zheng, D. L. Smith, D. R. Hinton, and P. S. Gill Vascular endothelial growth factor (VEGF) is an autocrine growth factor for VEGF receptor-positive human tumors Blood, September 15, 2001; 98(6): 1904 - 1913. [Abstract] [Full Text] [PDF]

				B. D. Smith, G. L. Smith, D. Carter, M. P. DiGiovanna, K. M. Kasowitz, C. T. Sasaki, and B. G. Haffty Molecular Marker Expression in Oral and Oropharyngeal Squamous Cell Carcinoma Arch Otolaryngol Head Neck Surg, July 1, 2001; 127(7): 780 - 785. [Abstract] [Full Text] [PDF]

				R. T.-P. Poon, S.-T. Fan, and J. Wong Clinical Implications of Circulating Angiogenic Factors in Cancer Patients J. Clin. Oncol., February 15, 2001; 19(4): 1207 - 1225. [Abstract] [Full Text] [PDF]

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