Mutation of p53 gene codon 63 in saliva as a molecular marker for oral squamous cell carcinomas

doi:10.1016/S1368-8375(00)00005-1

Oral Oncology

Volume 36, Issue 3, May 2000, Pages 272-276

https://doi.org/10.1016/S1368-8375(00)00005-1 Get rights and content

Abstract

The inactivation of tumor suppressor gene (TSG) is important during multistage carcinogenesis. The p53 TSG is frequently mutated in oral squamous cell carcinomas. These mutations can serve as very specific markers for the presence of tumor cells in a background of normal cells. In this study, 10 oral squamous cell carcinoma patients and 27 normal dental students were collected from Chung Shan Medical and Dental College Hospital, Taichung, Taiwan. Extractions of DNA from saliva were obtained. Exon 4 and intron 6 within the p53 gene were amplified with polymerase chain reactions (PCRs) followed by DNA sequence analysis. DNA sequence analysis of PCR products revealed that five of eight (62.5%) tumor saliva samples and five of 27 (18.52%) healthy saliva samples contained p53 exon 4 codon 63 mutations. These results were significantly different by using Chi-square test (P<0.05). The majority of the base substitutions were C deletions. Probable hot spots for the mutation were identified at exon 4 codon 63, which has not been observed before in head and neck cancers. Our study indicated that mutation of p53 codon 63 in saliva might be a molecular marker for oral squamous cell carcinomas. In addition, the amount of DNA recovered from saliva in most cases is sufficiently large and its quality suitable to enable PCR amplification which could be used in the search for mutations. The protocol described is rapid, cheap, and easy to perform, and may be useful for epidemiological studies for oral carcinogenesis.

Introduction

Cancer is considered to be a multi-hit process which involves a number of aberrant genetic events culminating in malignant transformation [1]. Oral cancer is the most frequent malignant tumor in the head and neck region, and about 80–90% is squamous cell carcinoma, which is the main pathologic type among all oral cancers [2]. The three major carcinogenic stimuli in the oral cavity, chemical, physical (radiation), and infectious (oncogenic viruses) agents, are mutagens which can change the structure of the genes and chromosomes by producing point mutations, deletions, insertions, or rearrangements [3]. These genetic alterations may result in distortions of either gene expression or biochemical function of gene products. Mutations may result in deregulation and constitutive activity, overproduction of a normal gene product, or alteration of the biochemical function of the gene product [4].

p53 gene encompasses 16–20 kb of DNA on the short arm of human chromosome 17 and contains 11 exons and is found as a tumor suppressor gene (TSG). TSGs are normal cellular genes when inappropriate inactivation leads to neoplastic transformation [5]. p53 genetic inactivation is, in most cases, attributed to its conformation mutations and allelic deletion [6]. The mutation or overexpression of the p53 gene is frequently found in squamous cell carcinomas of the head and neck [7], [8]. In addition, it plays an important role in the development of head and neck cancers.

Since oral squamous cell carcinoma is bathed in readily accessible secretions, we propose that exfoliated cells from the tumor might be detectable in saliva. The gene mutations in patients' primary tumor would serve as markers for these cells. Moreover, nearly half of primary head and neck carcinomas contain a mutation of the p53 gene, making this most commonly mutated gene known in this tumor type [9]. To test our hypothesis, we used a sensitive mutation-specific polymerase chain reaction (PCR) technique to determine the incidence and type of mutations of the p53 TSG of exfoliated oral cancer cells in saliva samples from patients with oral squamous cell carcinoma.

This study demonstrates the clinical application of new molecular techniques that can identify cancer cells among a large background of normal cells, and suggests possibilities for further oral cancer screening, diagnosis and follow-up based on the detection of genetic markers of cancer in the saliva.

Section snippets

Sample collection

Twenty-seven healthy volunteers were selected from dental students at Chung Shan Medical and Dental College, Taichung, Taiwan. They were non-smokers as well as non-betel quid chewers and had no clinical lesions in their oral mucosas. Ten patients with oral cancer were collected from the Department of Oral and Maxillofacial Surgery at the Chung Shan Medical and Dental Hospital, Taichung, Taiwan. Histology confirmed that all of these specimens were squamous cell carcinomas (Table 1). All these

Results

As shown in Fig. 1, the genomic DNA was successfully extracted from the exfoliated cells in saliva. Furthermore, the genomic DNA was successfully amplified through repeated cycles of denaturation, annealing and extension of primers for p53 exon 4 (Fig. 2) and intron 6 sites (Fig. 3).

After PCR product purification was performed, an ABIPRISM377 DNA autosequencer was used to detect DNA sequence. In our study, C deletion within p53 exon 4 at codon 63 was detected for five of eight (62.5%) of the

Discussion

Oral squamous cell carcinoma is the sixth most common cancer worldwide but third in the developing countries [10]. In spite of advances in surgery, radiotherapy and chemotherapy, the prognosis of this cancer is still very poor and has not changed over the past few decades. The overall 5-year survival rates are among the lowest of the major cancers at 30–40% [11]. Early diagnosis and early treatment are still the major determining factors in outcome [12].

Recently, studies have reported that

Acknowledgements

This work was supported by research grants from Chung Shan Medical and Dental College (CSMC 85-OM-B-001) and National Science Council (NSC 87-2314-B-040-016).

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Mutation of p53 gene codon 63 in saliva as a molecular marker for oral squamous cell carcinomas

Abstract

Introduction

Section snippets

Sample collection

Results

Discussion

Acknowledgements

Oral Oncology, European Journal of Cancer

Oral Oncology, European Journal of Cancer

Oral Oncology

Oral Oncology

The approaching era of tumor suppressor genes

Science

Mechanisms of carcinogenesis with particular reference to the oral mucosa

Journal of Oral Pathology

The molecular genetics of cancer

Science

Mutation in the p53 tumor suppressor gene: clue to cancer etiology and molecular pathogenesis

Cancer Research 1995

Tumor suppressor genes

Science