Overexpression of p 53 in Tumor-distant Epithelia of Head and Neck Cancer Patients Is Associated with an Increased Incidence of Second Primary Carcinoma 1

Second primary carcinoma is a peculiar feature of head and neck cancer and represents a form of treatment failure distinct from the recurrence of the primary tumor. Whether altered p53 expression in tumor-distant epithelia at the time of diagnosis is of clinical value as a biomarker for second primary carcinoma development has not been rigorously answered because of the lack of long-term follow-up studies involving a sufficiently large patient cohort. In this prospective study, we have investigated p53 expression in tumordistant epithelia and in the corresponding primary tumors of 105 head and neck cancer patients by immunohistochemistry on frozen sections. After a median follow-up of 55 months, the clinical course of disease parameters, i.e., local recurrences, lymph node and distant metastasis, incidence of second primary carcinoma, and survival, was evaluated. Overexpression of p53 in tumor-distant epithelia was found in 49 patients (46.7%), and it was independent of the p53 protein status of the primary tumor and of the tumor site, size, stage, and grading. Mucosal p53 overexpression was not associated with local primary recurrences, lymph node or distant metastases, or overall survival. Importantly, mucosal p53 overexpression, but not overexpression in the primary tumors, was significantly associated with an increased incidence of second primary carcinomas ( P 5 0.0001; Fisher’s exact test). When the times to second primary tumor occurrence were analyzed by the Kaplan-Meier method, the difference remained significant (P5 0.005; log rank test). We conclude that IHC staining for p53 overexpression in tumor-distant epithelia provides a simple and rapid tool to identify head and neck cancer patients at increased risk of developing second primary tumors. Because p53 overexpression in these epithelia in our patient cohort was specifically associated with second primary cancer but not with recurrences, at least a fraction of the second primary cancers appears to have resulted from genetic events in the mucosa (“field cancerization”). INTRODUCTION SCC of the head and neck is the sixth most common malignancy in the world, and the 5-year survival rate is one of the lowest among all cancers (1). Accumulation of mutations in oncogenes and tumor suppressor genes is a major molecular mechanism of tumor development (2). Among the tumor suppressor genes, inactivation of the p53 gene is one of the most frequent events (3). Overexpression of the p53 protein is frequently but not always associated with gene mutation and is regarded to be a promising candidate that might predict patient prognosis (4). Taking recent publications together, there is now good evidence for a prognostic impact of p53 mutations in head and neck cancer, i.e., they seem to be associated with an increased risk of locoregional treatment failure (5, 6). p53 DNA contact mutations appear to harbor a distinctly high malignant potential and to predict a poorer survival (7). Regarding p53 overexpression, Shin et al.(8), in their cohort of 69 patients with definitive local therapy (46% were stage I and II tumors), demonstrated a predictive value of p53 overexpression for shorter survival. However, other studies failed to confirm these results or revealed contradictory results (9–13). p53 overexpression not only occurs in 23–70% of all head and neck tumors (4) but also has commonly been found in dysplastic and nondysplastic premalignant epithelial lesions (9, 14–18). Even less is known about the clinical value of the observed p53 overexpression in these epithelia. Uhlmann et al. (15) performed a retrospective study on laryngeal cancer patients who previously underwent laryngeal biopsies without the diagnosis of cancer. Linear basal overexpression of p53 in premalignant lesions was found to be significantly associated with an increased risk of progression to cancer (15). In a prospective study, Galloet al. (16) have reported that simultaneous p53 and p16 INK4a alterations in premalignant laryngeal lesions have prognostic relevance in the progression to a malignant tumor. Thus, in the case of laryngeal precursor lesions, p53-overexpressing cells seem to have some malignant potenReceived 6/12/00; revised 11/20/00; accepted 11/20/00. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisementin accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This study was supported by the Wilhelm Sander-Stiftung, the Verein zur Förderung der Krebsforschung in Deutschland e.V., the Tumorzentrum Heidelberg/Mannheim, and the Forschungsförderungsprogramm der Medizinischen Fakultät Heidelberg. 2 To whom requests for reprints should be addressed, at HNO-Klinik, Molecular Biology Laboratory, University of Heidelberg, Im Neuenheimerfild 400, D-69120, Heidelberg, Germany. Phone: 49-6221-56-7278; Fax: 06221/564604; E-mail: Franz_Bosch@med.uni-heidelberg.de. 3 The abbreviation used is: SCC, squamous cell carcinoma. 290 Vol. 7, 290–296, February 2001 Clinical Cancer Research Research. on November 1, 2017. © 2001 American Association for Cancer clincancerres.aacrjournals.org Downloaded from tial. In oral premalignant lesions, the predictive power of p53 overexpression appeared even less pronounced than in laryngeal lesions. Ogdenet al. (17) have noted that p53 overexpression does not necessarily predict further malignant disease. Taking these studies together, it is clear that p53 overexpression will not turn out to be a definitive biomarker (indicating 100% risk) for second primary carcinoma (reviewed in Ref. 18). Whether p53 overexpression has reasonable predictive power at all has not been rigorously answered. For answering this question, longterm follow-up studies are required. For instance, on the basis of an actuarial rate of second primary cancer of 10% and on an 3-fold increase of this rate in a patient group with p53-positive, tumor-distant epithelia, a cohort size of 50 patients will only provide a study power of 50%. Therefore, the literature does not contain a study with a sufficiently large number of patients to answer this question. We have shown previously that in multiple anatomical sites within one cancer patient, histologically normal appearing tumor-distant epithelial cells can be found that express a mutated p53 protein (19). This finding, which was confirmed by Waridel et al. (20), has provided a possible molecular basis for the development of second (multiple) primary carcinomas. Here, we present a prospective long-term follow-up study involving 105 patients (including 15 patients from our previous work) with malignant head and neck tumors. We have focused on the analysis of p53 protein expression in tumor-distant epithelia at the time of surgery of the primary tumor and have compared this with clinicopathological parameters, including the occurrence of second primary carcinoma. Immunohistochemical staining of tissue sections is a simple, rapid, and reliable method to assess p53 protein expression and can be performed according to standard protocols in many clinical laboratories. The size of our patient cohort was estimated to be sufficiently large to answer whether p53 overexpression is associated with second primary carcinomas. Our results show that it is indeed possible, using this simple assay, to identify patients with higher and lower risk for second primary SCCs of the head and neck. PATIENTS AND METHODS Patients and Tissue Samples. Clinicopathological parameters from patients with head and neck cancer were obtained from patient records and the statements of the pathology department. Only patients from whom fresh-frozen biopsies could be obtained and who were regularly seen at follow-up examinations were recruited. The enrollment period started from May 1990 and ended in December 1998. Inclusion criteria were SCC only (excluding skin cancer) and patients presenting with primary tumors (excluding patients with recurrent tumors and patients with secondary tumors who had been treated with radiation and/or chemotherapy) at the Department of OtoRhino-Laryngology of the University of Heidelberg. Accuracy of the clinical data was validated by two independently reviewing investigators, who were unaware of the results of p53 staining. The data collected included age, gender, primary tumor site (oral cavity, oropharynx, larynx, hypopharynx, or other), tumor size (T1–4), lymph nodal status (N 0–3), American Joint Committee on Cancer Stage (stages I–IV), histological grading (well, moderately, poorly, and undifferentiated), and cause of death. In follow-up, recurrences of primary tumor and occurrences of lymph node metastases, distant metastases, and second primary tumors were recorded. For the diagnosis of second primary tumors, a modification of the criteria of Warren and Gates was used (see Ref. 21). Only second primary tumors occurring in the aerodigestive tract were enrolled in this study. All tissues were collected during the operation and immediately snap-frozen in 2-methyl-butane, precooled in liquid nitrogen, and stored until use at 280 C. One hundred fifty-one snap-frozen, tumor-distant epithelial tissues were available for study. Precise information on the anatomical location was available in 113 cases (the other biopsies were merely classified as umor-distant). The tumor-distant epithelia were derived from the site adjacent to the tumor (e.g., oropharyngeal mucosa in case of a larynx carcinoma and hypopharyngeal mucosa in case of an oropharynx carcinoma) or from the contralateral subsite in case of oral cavity and larynx tumors (see Table 2). These small biopsies had a minimum distance of 4 cm to the margin of the tumor and were histopathologically tumor free. Epithelia of 63 patients who underwent operation for other reasons than malignancy were collected for comparison. Immunohistochemistry. Immunohistochemical staining of the frozen sections wit