p53 tumor suppressor gene as a clonal marker in head and neck squamous cell carcinoma: p53 mutations in primary tumor and matched lymph node metastases

In order to define the diagnostic value of p53 tumor suppressor gene as a c lonal marker in head and neck squamous cell carcinoma (HNSCC), we investiga ted p53 mutations in primary tumors (PT) and matched lymph node metastases (LNM); the underlying question being whether differentiation between metast atic disease of a known PT or (a metastasis of) a synchronous or metachrono us second tumor is possible by means of p53 sequencing-based mutation analy sis. In 15 PT, the p53 status was analyzed, following RNA isolation, cDNA s ynthesis and polymerase chain reaction amplification, by direct sequencing full-length mRNA. Mutations thus found were confirmed by DNA sequencing ana lysis of the corresponding exon in the PT. When RNA isolation was defective , DNA sequencing analysis of exons 1 through 11 was performed. In the match ed LNM, DNA analysis of the corresponding exon was performed to prove the p resence of the same p53 mutation. In the event of small clones not detectab le by direct sequencing, an oligo ligation assay was developed to detect a specific mutation. The presence Of germline mutations was excluded by DNA s equencing analysis of the corresponding exon of peripheral blood leucocytes . In 14 PT (94%), a mutation was identified. In one PT, no p53 mutation cou ld be identified either after full-length mRNA sequencing or after sequenci ng exons 1 through 1 1. In all cases of PT and matched LNM, the mutations p roved to be identical. We conclude that p53 mutations develop in carcinogen esis before metastases occur and are maintained during metastasis. Conseque ntly, p53 may serve as a clonal marker not susceptible to change during tum or metastasis. This merits further exploration of the application of p53 mu tation analysis in differentiating between metastatic disease from a known PT versus a metastasis of another second PT. (C) 1999 Elsevier Science Ltd. All rights reserved.