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.
Department of Oto-Rhino-Laryngology, Head and Neck Surgery, University of Leipzig, Liebigstrasse 18a, 04103 Leipzig, Germany.
Anette
A
Weber
Ulf
U
Bellmann
Friedrich
F
Bootz
Christian
C
Wittekind
Andrea
A
Tannapfel
D014408:Biomarkers, Tumor / Q000032:analysis; D002294:Carcinoma, Squamous Cell / Q000737:chemistry / Q000235:genetics* / Q000556:secondary; D019941:Cyclin-Dependent Kinase Inhibitor p16 / Q000032:analysis / Q000235:genetics*; D019175:DNA Methylation; D017931:DNA Primers / Q000737:chemistry; D004273:DNA, Neoplasm / Q000032:analysis; D016158:Genes, p53; D006258:Head and Neck Neoplasms / Q000737:chemistry / Q000235:genetics* / Q000473:pathology; D006801:Humans; D007150:Immunohistochemistry; D009154:Mutation; D009364:Neoplasm Recurrence, Local; D016609:Neoplasms, Second Primary / Q000737:chemistry / Q000235:genetics* / Q000473:pathology; D012333:RNA, Messenger / Q000032:analysis; D012334:RNA, Neoplasm / Q000032:analysis; D020133:Reverse Transcriptase Polymerase Chain Reaction; D025803:Tumor Suppressor Protein p14ARF / Q000032:analysis / Q000235:genetics*
D014408:Biomarkers, Tumor; D019941:Cyclin-Dependent Kinase Inhibitor p16; D017931:DNA Primers; D004273:DNA, Neoplasm; D012333:RNA, Messenger; D012334:RNA, Neoplasm; D025803:Tumor Suppressor Protein p14ARF