Oncogene (1997) 14, 811 ± 818 1997 Stockton Press All rights reserved 0950 ± 9232/97 $12.00 Alterations of the p16 gene in head and neck cancer: frequency and association with p53, PRAD-1 and HPV Andrew F Olshan1,2, Mark C Weissler2, Hong Pei1, Kathleen Conway1, Steven Anderson3, Daniel B Fried1 and Wendell G Yarbrough2 1Department of Epidemiology, School of Public Health, University of North Carolina, Chapel Hill, North Carolina; 2Division of Otolaryngology/Head and Neck Surgery, School of Medicine, University of North Carolina, Chapel Hill, North Carolina; 3Department of Molecular Biology and Pathology, Laboratory Corporation of America, Research Triangle Park, North Carolina, USA Alterations, especially homozygous deletions, of the suppressor gene (Brachman, 1994). Recently, the putative tumor suppressor gene, p16 (p16INK4A, MTS1, cyclin-dependent kinase inhibitor, p16 (p16INK4A, CDKN2) have been found in tumor cell lines from a MTS1, CDKN2), has been proposed as the tumor variety of neoplasms. Recent studies have reported suppressor gene at 9p21 (Serrano et al., 1993, 1996; frequent p16 gene deletions in cell lines from squamous Kamb et al., 1994; Nobori et al., 1994; Lukas et al., cell carcinomas of the head and neck (SCCHN), 1995a; Koh et al., 1995; Spillare et al., 1996). Related although the prevalence of alterations was variable in CDK-inhibitors have also been isolated including primary tumors. This study determined the prevalence of p15INK4b, p18INK4C and p19INK4d (Sherr and Roberts, point mutations and deletions of the p16 gene in 33 1995). Further, the p16INK4a locus yields two transcripts SCCHN. In addition, the association of p16 gene one encoding p16INK4a and another encoding p19ARF alterations and abnormalities of p53, PRAD-1 (cyclin (Quelle et al., 1995). All of these genes participate D1), and the presence of human papillomavirus (HPV) either directly or indirectly in the control of the cell was examined. We found an overall prevalence of p16 cycle. alterations of 36% (nine deletions, three single base The transition from G1 to S in the cell cycle relies on substitutions, including one polymorphism). Seven tumors a coordinated interaction of several gene products (of 29, 24%) had an alteration of p16 and p53; ®ve (of including the retinoblastoma protein (Rb), cyclin D1, 33, 15%) had alterations of p16 and PRAD-1; three (of and its catalytic partners CDK4 and CDK6, and the 29, 10%) had alterations of all three genes. In addition, cyclin-dependent kinase inhibitor, p16 (Peter and of the ®ve tumors with human papillomavirus detected, Herskowitz, 1994; Grana and Reddy, 1995; Yeudall only one also had a p16 gene alteration. The results and Jakus, 1995). Loss of control of the G1 to S indicate a potentially important role for the p16 gene in transition is a common defect observed in many head and neck tumorigenesis. In addition, the presence of dierent tumor types and can occur through a variety tumors with multiple somatic gene alterations suggest a of mechanisms (Hartwell and Kastan, 1994; Kamb, possible interaction in the dysregulation of the cell cycle. 1995). Viruses that are associated with tumors, such as the human papillomavirus (HPV) and SV40, produce Keywords: head and neck neoplasms; mutation; p53; proteins that inactivate Rb and p53. Also, mutations p16INKA4; HPV; PRAD-1 and/or deletions of RB, p53, and p16 and ampli®cation and overexpression of cyclin D1 are relatively common events in a wide range of tumors. Recently, evidence for transcriptional repression of p16 through promotor Introduction methylation has been observed in all major solid tumor types, suggesting another mechanism through which Squamous cell carcinoma of the head and neck loss of control of the G1-S transition can occur (Merlo (SCCHN, including oral cavity, pharynx and larynx) et al., 1995). It has been suggested that dierent genes provides an excellent model for understanding the related to the control of the cell cycle represent carcinogenic process due to the de®ned risk factors alternate targets and that functionally inactivating (tobacco and alcohol consumption) and emerging any one component will have the same consequence molecular characteristics. Cytogenetic, allelotyping, of loss of cell cycle control. Little research has been loss of heterozygosity, and gene ampli®cation studies done to evaluate alternate pathways by examining the have implicated 11q as the site of a putative oncogene, interrelationship among abnormalities in cell cycle and 17p, 9p and 3p as sites of putative tumor genes in tumors. The relationship betwen p16 and suppressor genes in SCCHN (Cowan et al., 1992; other genes, including HPV has not been directly Nawroz et al., 1994; Van der Reit et al., 1994). investigated in SCCHN. Molecular studies have identi®ed candidate genes for Examination of primary tumors and cell lines for these regions, namely cyclin D1 (PRAD-1, CCND1) as alterations of the p16 gene has yielded con¯icting the protooncogene and p53 as a putative tumor results. Initial reports indicated a high frequency of homozygous deletions of p16 in a variety of cell lines (Kamb et al., 1994; Nobori et al., 1994). In addition, Correspondence: AF Olshan point mutations were identi®ed in cell lines without Received 14 June 1996; revised 18 October 1996; accepted 18 October deletions. Other studies using primary tumors reported 1996 a much lower prevalence of p16 deletions and p16 gene alterations and head and neck cancer A F Olshan et al 812 mutations, suggesting that tumor cells might acquire a p16 defects as a consequence of forming established 12345678 lines (Carins et al., 1994; Spruck et al., 1994). More GAPDH recent studies suggest that p16 defects are not an p16 artifact of establishing cell lines because they occur in a high percentage of certain types of primary carcinomas (Mori et al., 1994; Zhou et al., 1994; Caldas et al., p16 1994; Schmidt et al., 1994; Carins et al., 1995). Two CPT analyses of primary head and neck tumors have reported a prevalence of point mutations of 7% and 10%, con®rming that the loss of p16 function more commonly occurs through deletion (Carins et al., 1994; Zhang et al., 1994). Two recent reports indicated a b higher prevalence of p16 gene alterations in head and 123456 neck tumors 25% (16/65; all deletions; Carins et al., GAPDH 1995) and 19% (7/36; six deletions; Lydiatt et al., p16 1995). None of the studies of SCCHN evaluated the relationship between p16 gene alterations and the p16 presence of alterations of other cell cycle associated CPT genes. In this study, we determined the prevalence of p16 0 20 40 60 80 100 somatic alterations in 33 SCCHN tumors and have % Normal DNA examined the association of p16 mutation or deletion Figure 1 Representative deletion analysis of p16 gene in head with aberrations of other tumor suppressor genes and and neck tumors. (a), PCR products from six tumors and two oncogenes, and with the presence of human papillo- control samples with two reference genes, GAPDH and CPT. mavirus (HPV). Our results provide additional Lanes 2, 4, 5, deletions of the p16 target gene. Lane 7, sample of normal tonsil tissue. Lane 8, cell line with p16 homozygous evidence for the role of the inactivation of p16 in the deletion (K562). The estimated percentage of contaminating development of head and neck cancer. normal cells and ratio of the intensity of the target gene band to the reference gene (CPT) was: lane 1 (70%, 1.04), 2 (25%, 0.24), 3 (60%, 1.03), 4 (70%, 0.64), 5 (60%, 0.63), 6 (60%, 1.05), 7 (100%, 1.00), 8 (0%, 0.00). (b) Comparison PCR using a mixed Results DNA template from human foreskin ®broblast and a human leukemia cell line (K562) with a known homozygous deletion of Among the 33 tumors examined, a total of 12 (36%) the p16 gene and GAPDH and CPT as reference genes. The had either a deletion (n=9) or single base substitution percentage of normal DNA mixed in the template is shown at the in exon 2 of the p16 gene (n=3). Using dierential bottom of the lanes. The ratio of the intensity of the target gene band to the reference gene (CPT) band for each sample was: lane PCR, independently coampli®ed each time with a 1 (0.00), 2 (0.31), 3 (0.53), 4 (0.69), 5 (0.78), 6 (1.01) dierent set of reference genes (PGR/d-IFN, GAPDH/CPT), we detected p16 deletions in 9 (27%) of the tumors (Figure 1 and Table 1). Figure 2 shows an example of selected mutations. tumors had a p16 base change, did not show any p16 Four mutations were identi®ed in three tumors mutations or deletions. Although there were some including an A4T transversion (GAC4GTC, dierences, no signi®cant association was found Asp4Val) at codon 76 (case #2300), a G4A between the presence of a p16 mutation and SCCHN transition (GCG4ACG, Ala4Thr) at codon 140 site (P=0.74), recurrence status (P=0.43), or history (case #4165), and a tumor (case #3515) with two of preoperative radio- or chemotherapy (P=0.42). G4T transversion mutations, one at codon 79 Additionally, there was no relation between p16 (CGG4CTG, Arg4Leu), the other at codon 80 alterations and Tumor-Node-Metastasis (TNM) stage (GAG4TAG, Glu4Stop). Mutations at codon 76 classi®cation (P=0.47). have been reported in a previous study of head and We were also able to successfully screen 29 of the 33 neck tumors and in a study of esophageal cancer SCCHN tumors for p53 mutations using SSCP and (Spruck et al., 1994; Mori et al., 1994). In both cases, direct sequencing. Twelve of 29 (41%) tumors were the sequence change was a transition from GAC to found to have a mutation of the p53 gene and seven of AAC; in our study the change was a transversion these 12 also had a p16 alteration (®ve deletions and from GAC to GTC.