INK4a-ARF alterations and p53 mutations in primary and consecutive squamous cell carcinoma of the head and neck.

BACKGROUND: The INK4a-ARF (CDKN2A) locus, located on chromosome 9p21, encodes two functionally distinct tumor suppressor genes, p14(ARF) and p16(INK4a), that play active roles in the p53 and Rb tumor suppressive pathways, respectively. We analyzed the alterations of p14(ARF), p16(INK4a) and p53 to study the contribution of each pathway in tumorigenesis of 29 patients with primary and consecutive (second primary) squamous cell carcinoma of the head and neck (HNSCC), with a total of 68 carcinomas. METHODS: After microdissection, the DNA of 29 primary and 39 consecutive squamous cell carcinomas was analyzed for INK4a-ARF inactivation and p53 mutation by means of DNA sequence analysis, methylation-specific polymerase chain reaction (MSP), restriction-enzyme-related polymerase chain reaction (RE-PCR), multiplex RT-PCR and immunohistochemistry. In addition, microdeletions of p14(ARF) and p16(INK4a) were assessed using differential PCR. RESULTS: Altogether inactivation (methylation, loss of heterozygosity and mutation of exon 1beta) of p14(ARF) was found in 29 of all 68 (43%) carcinomas, with a significant difference in primary [8 of 29 (28%)] relative to second primary carcinomas [21 of 39 (54%)]. Methylation of p16(INK4a) occurred in 22 of 68 (32%) carcinomas with an even distribution among primary and consecutive tumors. Only two (secondary) carcinomas showed simultaneous promoter methylation of p14(ARF) and p16 (INK4a). Mutations of p53 were found in 32 of 68 HNSCCs (44%), evenly distributed among primary and recurrent carcinomas. p14(ARF) alterations showed no relationship to p53 mutations. CONCLUSIONS: Our data indicate that the INK4a-ARF-/p53 pathway was disrupted in 58 of 68 (84%) primary and recurrent tumors, either by p53 mutations or by INK4a-ARF inactivation. p14(ARF) methylation occurred independently of p16(INK4a) alterations and showed no correlation to p53 mutations. The significantly higher rate of p14(ARF) alterations in recurrent (respective second primary) carcinomas suggests a further acquired genetic aberration during the development of the recurrent carcinomas.

Expression of p53 and its homologues in primary and recurrent squamous cell carcinomas of the head and neck.

The tumour-suppressor protein p53 belongs to a family that includes 2 structurally related proteins, p63 and p73. Because of their structural homology, it has been hypothesized that both homologues serve as "spare mechanisms" in p53 mutations to regulate the cell cycle by inducing apoptosis. We investigated the mutational and protein expression status of p53 in correlation to its homologues, p73 and p63, in primary and recurrent squamous cell carcinomas of the head and neck (HNSCC) and corresponding nonneoplastic mucosa. Expression and mutation of p53 and its homologues p63 (including the 2 major isotypes TAp63 and DeltaNp63) and p73 was examined by direct DNA sequencing and immunohistochemistry in 29 primary and 39 recurrent (secondary) HNSCCs after microdissection. Our results were correlated with pathohistologic stage and grade. p53 mutations were detected in 32/68 (47%) carcinomas of 17 patients, with a discordant mutation pattern of primary and consecutive tumours in all cases. Positive immunostaining for p63 was found in 55/68 (81%) carcinomas of 29 patients. Immunohistochemistry revealed p73 protein expression in 32/68 (47%) tumours. In normal mucosa, p63 and p73 were expressed in 40/68 (59%) and 12/68 (18%) cases, respectively. We failed to detect specific mutations of p73 or p63 in primary and recurrent carcinoma of the head and neck. p73 and p63 were rarely mutated in HNSCC, but both were expressed in a subset of tumours. The lack of correlation between p73/p63 and p53 protein expression suggests that neither p73 nor p63 can replace p53 when it is mutated.