Published in Cancer Detection and Prevention 2001; 25(2):109-116.

Sensitive Detection of p53 Mutation Analysis by Direct Sequencing and Multisequence Analysis

Erik H. Rozemuller, PhDa, Arvid Kropveld, PhDb, Ellen Kreyvelda, Fredriek G.J. Leppersa, Karen C. Scheidela, Piet J. Slootweg, MD, PhDa and Marcel G.J. Tilanus, PhDa

Departments of a Pathology, Section of Molecular Immuno Pathology, and b Otorhinolaryngology, University Medical Center, Utrecht, The Netherlands.

Address all correspondence and reprint requests to: Erik H. Rozemuller, PhD. Department of Pathology, Section of Molecular Immuno Pathology, University Medical Center, Utrecht, P.O. Box 85500, 3508GA Utrecht. The Netherlands.

ABSTRACT: In a wide variety of tumors, p53 mutations may have prognostic and diagnostic value. However, mutational screening methods often are restricted to the core domain and, therefore, do not detect all mutations. We improved existing sequencing-based mutation analysis methods consisting of direct sequencing of all exons of the p53 gene and RNA. Multisequence analysis software was developed and applied to increase the sensitivity of mutation identification. Multisequence analysis compares a large number of sequences and identifies profiles with additional small peaks, potentially indicating a mutation. Concordance between blood and tumor sequences indicates polymorphism, whereas discordance indicates a mutation. We could detect mutations with a limit of approximately 5% to 10% mutated DNA. In our ongoing studies, we applied sequencing-based mutation analysis for more than 50 patients with head and neck squamous cell carcinoma and identified mutations in more than 95% of all tumors. We encountered some differences between the previously published reference p53 sequence and all our sequences and identified polymorphism in six regions.

KEY WORDS: p53, multisequence analysis, mutation, polymorphism, sequencing.