Loss of Heterozygosity and Mutation Analysis of the p 16 ( 9 p 2 l ) and p 53 ( l 7 pl 3 ) Genes in Squamous Cell Carcinoma of the Head and Neck 1

We analyzed ablelic loss at the p13 gene (l7pl3) and at chromosome region 9p21 in 35 primary head and neck squamous cell carcinomas. Loss of heterozygosity (LOH) at p53 and 9p2l was found in 50 and 75% of informative cases, respectively. LOH at the p53 gene did not increase significantly with tumor stage, but was more frequent in moderately and poorly differentiated tumors than in well-differentiated tumors. LOH plus mutation or homozygous deletion of p53 was limited to advanced stage and poorly differentiated tumors. Allelic loss at 9p2l is frequent in early stage head and neck squamous cell carcinoma and is not significantly associated with LOH at p5.3. The second exon of the p16/MTSI/CDKN2 gene was found to be homozygoushy deleted in 1 of 19 cases showing LOH at 9p2l, but direct sequencing did not show mutations in the remaining 18 cases. This suggests that p16 plays a limited role in the development of head and neck squamous cell carcinoma. INTRODUCTION HNSCC4 is one of the most common types of cancer in the world. More than 10,000 Americans die each year from HNSCC (1). Its 5-year survival rate has not changed in recent years, and is still one of the lowest among the prevalent cancers. HNSCC is strongly associated with tobacco and alcohol consumption (2). Little is known about the genetic events involved in its progression, although some genes have been reported to contribute significantly (3). Mutations have been found in the oncogene Ha-ras (4). Overexpression of cyclin D has been reported to be associated with aggressive behavior of HNSCC Received 2/17/95; revised 4/4/95; accepted 5/4/95. 1 This work was supported by Grant FISS 94/1421 (E. C.). 2 Recipient of a fellowship of the FiCYT. 3 To whom requests for reprints should be addressed. 4 The abbreviations used are: HNSCC, head and neck squamous cell carcinoma; TSG, tumor suppressor gene; LOH, loss of heterozygosity; SSCP, single-strand conformation polymorphism; cdk4, cychin-dependent kinase 4; VTR, variable tandem repeat(s). (5-7). AJlebotyping using DNA microsatellite markers representative of all human chromosomes have found regions of common LOH (8, 9), an event associated with the inactivation of TSGs. To date, the highest mutation rate for any gene in the HNSCC is that of p53 (chromosome l7pl3; Refs. 10-13), which is also mutated in most human cancers (14). Mutations of the p.53 gene in HNSCC resembles those found in the squamous cell lung carcinoma. p.53 is known to be a TSG (15) that encodes a nuclear protein of 53,000, which acts as a transcription factor (16-19) through sequence-specific DNA binding (20, 21). Mutations in p53 abrogate its transactivating activity, leading to uncontrolled cell growth (22). Approximately 90% of the p53 mutations are located in exons 5-9. These exons encode the surface of the protein, and amino acids at this region are directly involved in DNA binding (23). Recent studies have described the presence of homozygous deletions in multiple tumor-derived cell lines (24) and in primary melanoma (25, 26), pancreatic carcinoma (27), and esophageal squamous cell carcinoma (28) on another TSG, p16/ MTSJ/CDKN2, mapped to chromosome 9p2l. The protein binds to cdk-4, preventing it from forming a complex with cyclin D. The cyclin D/cdk-4 complex allows the cell cycle to progress through G1, thus permitting cell division. If cdk-4 is overexpressed or expressed at the wrong moment, then pitS would exert its role as a TSG by binding cdk-4 and inhibiting cell growth (29). TSGs are generally inactivated by the boss of one copy and inactivation ofthe remaining copy by point mutation or deletion. In an attempt to determine the robe of p53 and p16, we have analyzed several microsatellite markers that are intragenic or closely linked to these genes, and searched for mutations at these genes in 35 unselected HNSCCs. MATERIALS AND METHODS Patients and DNA Extraction. Paired tumor and blood samples were collected from 35 patients in the operating room immediately after operation. For tumor DNA extraction, the fresh tumor tissue was isolated by microdissection, and specimens containing more than 70% neoplastic cells were placed at 37#{176}C for 24 h in lysis solution containing SDS and proteinase K. DNA was obtained after phenol-chloroform extraction and ethanol precipitation (30). DNA from WBCs was obtained in accordance with a previously described protocol (31). The 35 samples were from the pharynx (20 samples), larynx (13 samples), and mouth floor (2 samples). Histologically, 13 were well-differentiated, 12 were moderately differentiated, and 10 were poorly differentiated tumors. In reference to the tumor stage, 1 neoplasm was stage I, 9 were stage II, 15 were stage III, and 10 were stage IV tumors. Research. on October 31, 2017. © 1995 American Association for Cancer clincancerres.aacrjournals. org Downloaded from