Elsevier

Human Pathology

Volume 36, Issue 2, February 2005, Pages 187-194
Human Pathology

Original contribution
p63 overexpression associates with poor prognosis in head and neck squamous cell carcinoma

https://doi.org/10.1016/j.humpath.2004.12.003Get rights and content

Summary

p63 belongs to a protein family that includes 2 structurally related proteins, p53 and p73. The aim of this study was to investigate the biologic role of p63 in oral tumorigenesis and its possible role as prognostic marker in oral cancer. Ninety-four cases of oral squamous cell carcinoma and 10 cases of normal mucosa were analyzed for p63 expression by immunohistochemistry. Normal oral mucosa showed a basal and parabasal expression of p63. Five (5.3%) cases of oral cancer showed less than 10% of positive tumor cells; in 33 (35.1%) cases the positive tumor cells comprised between 10% and less than 30%, in 36 (38.3%) cases the positive tumor cells comprised between 30% and less than 50%, and in 20 (21.3%) cases the positive tumor cells were more than 50%. There was also a statistically significant correlation between p63 expression and tumor differentiation: p63 expression was amplified in poorly differentiated tumors (P < .05). When analyzed for prognostic significance, patients with perineural infiltration had poorer survival rates than the group with no perineural infiltration (P < .05) and patients with increased p63 expression had poorer survival rates than the group with reduced p63 expression (P < .05). The statistical analysis showed no significant correlation between p63 expression, sex, age, tumor size, staging, recurrence, and metastasis. Cases with diffuse p63 expression were more aggressive and poorly differentiated and related to a poorer prognosis. These data suggest that p63 expression may be useful to identify cases of oral squamous cell carcinoma with more aggressive and invasive phenotype providing novel diagnostic and prognostic information on individual patient survival with oral cancers.

Introduction

The TP53 tumor-suppressor gene encodes a multifunctional DNA-binding protein important in cell cycle and cell death regulation, and is the most frequently altered gene in human cancers [1], [2]. Despite the central role of TP53 in tumorigenesis, no related genes were found for 20 years [3]. But, recently, 2 new members of the TP53 gene family, named p73 and p63, have been identified [4], [5]. Each gene is approximately 65 kilobase; they show a high degree of similarity in exon/intron organization [6], [7], [8]. The p63 gene is located at the 3q27-29 region and contains 15 exons [4]. Unlike the unique and alternative, but infrequently used, splicing of the TP53 gene, both p63 and p73 give rise to an array of multiple protein isoforms due to differential mRNA splicing and to alternative promoter usage [9], [10], [11]. The p63 gene undergo multiple C-terminal splicing [3], but, among the high number of spliced isoforms found at the RNA level, 3 of them are consistently found at the protein level [9], [12]. The 3 spliced forms of p63 produced are termed α, full structure; β, splicing of exon 13; and γ, splicing from exons 10 to 15 [9], [12].

Human TP53 gene has a single promoter that encodes a single protein of 393 amino acids [3]. In contrast, p63 gene expression is regulated by the occurrence of 2 different promoters that leads to the production of 2 different classes of proteins [3], [4], [9], [13]. The P1 promoter is located in the 5′ untranslating region (UTR) upstream of a noncoding exon 1 [3] and leads isoforms showing the acidic N-terminal transactivation (TA) domain (TAp63) [4], [9], [14]. The P2 promoter (cryptic promoter) [16] is located within the 23-kilobase-spanning intron 3 [3] and encodes isoforms lacking the N-terminal transactivation domain (ΔNp63) [4], [9], [10], [14].

The p63 gene plays an essential role in epithelial development and the proliferation of limb and craniofacial structure [15]. Studies with murine model have revealed that p63 knockout mice are born alive but display severe deformation of limbs and profound defects in craniofacial development, as well as in differentiation of tissues with stratified epithelium including skin, oral cavity, esophagus, breast, urothelium, and prostate [13], [16]. A similar pattern was observed in patients with the ectrodactyly, ectodermal dysplasia, and facial clefts (EEC) syndrome, an autosomal dominant disorder characterized by ectrodactyly, ectodermal dysplasia, and facial clefts, where unrelated patients are found to have dominant heterozygous p63 mutations [3], [9].

As previously said, the p63 gene encodes 6 different isoforms: 3 with the transactivating domain (TA) and 3 lacking the N-terminal domain (ΔN). Several lines of evidences suggested that these isoforms exert opposite biologic properties. The TAp63 forms are capable of transactivating p53 target genes also inducing apoptosis, whereas ΔNp63 forms behave in a dominant negative fashion toward their own TA proteins and toward p53 in vivo in the mouse and in transfected human cells [3], [17]. Strikingly, squamous cell carcinoma of the skin and other organs produce high levels of ΔNp63 [3]. Furthermore, the chromosomal p63 locus is not subject to loss but to gain in this cancer type and therefore suggests some oncogenic function for p63 [3], [9].

The frequency of oral squamous cell carcinoma (OSCC) is rapidly increasing and, in particular, it constitutes the most frequent malignant tumor of the oral cavity. The incidence of metastasis depends on the degree of cellular differentiation, degree of invasion, and site of the primary tumor. However, the clinical behavior of this tumor is difficult to predict based on classic histopathologic parameters alone. Biologic markers that can identify the lesions with an aggressive phenotype and worse prognosis need to be identified.

Because of the almost restricted expression of p63 in epithelial cells, its infrequent mutation, as well as its overexpression in various solid tumors, indicating an oncogenic role in the regulation of proliferation and differentiation in premalignant and malignant lesions of epithelial origin, a considerable interest has recently been focused on p63.

To understand the biologic role of p63 in oral tumorigenesis, the present study analyzed the p63 expression in oral cancers by immunohistochemistry, correlating its expression with clinicopathologic features. Moreover, the ability of p63 to identify cases of OSCC with more aggressive and invasive phenotype providing novel diagnostic and prognostic information on individual patient survival was evaluated.

Section snippets

Selection of cases

The study population consisted of 60 men and 34 women with a mean age of 65 years (range, 11-92 years); 41 cases were stage I, 17 stage II, 13 stage III, and 23 stage IV. All patients were analyzed for survival rates. Survival was calculated from the date of surgery to the date of the latest clinical follow-up or death by disease. Patients who died of postoperative complications were excluded from the study.

None of the patients had been treated previously. They received surgical treatment with

p63 expression in normal oral mucosa

Normal human oral mucous epithelium had a basal and parabasal pattern of p63 expression. The labeling was only nuclear, with nuclei showing an intense staining, stronger in the basal layer with respect to the parabasal layer (with nuclei of the parabasal layer showing only a faint staining). Generally, keratinocytes of suprabasal layers were not immunolabeled by anti-p63 antibody, although a slight expression of p63 was recorded in some areas. Thus, normal epithelium included around 10% of

Discussion

In the current investigation, 10 paraffin-embedded specimens of healthy oral mucosa were obtained from patients who had undergone routine oral surgical procedures and analyzed for immunohistochemical expression of p63. The results showed that in normal human oral mucosa, p63 had a restricted and precise pattern of expression. Indeed, we found a nuclear staining in all cases of normal epithelium confined at the basal and, less evident, at the parabasal layers. In our study, findings were

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