Three Discrete Regions of Deletion at 3p in Head and Neck Cancers 1

Alteration of the short arm of chromosome 3 is one of the most consistent cytogenetic abnormalities found in human head and neck cancers. These alterations, composed of translocations and deletions, have been associated with the presence of a tumor suppressor gene(s), but no clear evidence of the location of this presumptive gene(s) was available. We performed a molecular analysis of the 3p region using a polymerase chain reaction-based approach. Twenty-eight of the 38 cases analyzed (74%) showed the presence of single or multiple areas of allelic loss. Three commonly deleted regions, tentatively mapped to 3p24-ter, 3p21.3, and 3pl4--cen, were identified. Our results suggest that at least three oncosuppressor genes mapping on 3p may be involved in head and neck cancer development and support a common oncogenic pathway with squamous cell lung cancer, for which a similar pattern of 3p deletion has been described recently. I N T R O D U C T I O N Cancer is considered to be a multistage process involving a number of aberrant molecular events culminating in malignant transformation. Considerable evidence demonstrates that genetic alterations in both dominant oncogenes and oncosuppressor genes are responsible for human cancer development. The identification of such phenomena may be useful in deepening the knowledge of the biological features of neoplastic cells and, in particular, it may allow the identification of new diagnostic and prognostic markers, thus providing a basis for more appropriate therapeutic approaches. With regard to HNSCC, 4 basic studies have been largely neglected mainly because of the relatively limited frequency of this neoplasm. However, the incidence of this type of tumor is expected to rise as a result of the increasing number of female and adolescent smokers and as a consequence of environmental pollution (1, 2). Moreover, despite the great emphasis on early diagnosis and the efforts to improve surgical, pharmacological, and radiation treatment management, about 50% of HNSCC patients do not survive for more than 5 years after diagnosis (3). The growing epidemiological problem and the lack of progress in head and neck oncology emphasizes the need for basic studies on the molecular biology of HNSCC. Cytogenetic studies have revealed that the short arm of chromosome 3 (3p) is frequently affected by chromosomal rearrangements and deletions in HNSCC (4--6). In addition, HNSCC cell lines show an excess of homozygosity for 3p RFLP markers (7), which is considered synonymous with deletion phenomena. These data suggest that the knocking out of one or more genes located at 3p may play a role in the pathogenesis of HNSCC. In fact, according to Knudson's hypothesis (8), in cancer cells, mutation of a tumor suppressor gene is frequently accompanied by loss of the remaining allele and flanking regions. Consequently, analysis of allelic losses may allow the identification of chromosomal regions harboring tumor suppressor genes. The present study was performed with the aim to evaluate the role of 3p allelic losses in HNSCC and to define the minimal chromosomal regions of 3p which, being specifically involved in the phenomenon, suggest the presence of tumor suppressor genes responsible for tumor development. M A T E R I A L S AND M E T H O D S Samples. Matched tumor and corresponding normal mucosa were obtained from 38 patients with primary HNSCC. No patient had been treated with chemoor radiotherapy prior to surgery. All tissues were frozen in liquid nitrogen immediately after surgery and stored at -80~ until extraction of DNA. Frozen tissues were powdered with a cell dismembrator. Genomic DNA extraction was performed with a DNA extractor according to the manufacturer's instructions (Applied Biosystem, Inc., Foster City, CA). Analysis of LOH Using RFLP and Microsatellite Polymorphisms. Analysis of 3p allelic deletions was performed using a PCR-based approach. Polymorphic loci and relative amplification primers are listed in Table 1. Chromosomal position and sequential order on the chromosome are according to the criteria of Jones and Nakamura (9), Jones et al. (10), and Yokoyama et al. (11). LOH for the loci THR-B, DNF15S2, D3S32, D3S2, and D3S30 was evaluated with a PCR-based RFLP approach using the primers and restriction enzymes listed in Table 1. PCRs were performed in a 50-p,l reaction volume as described previously (12). Reactions were heated to 94~ for 3 min and then cycled 40 times. Each cycle consisted of denaturation at 94~ for 1 min, annealing at the appropriate temperature for 1 min (see Table 1), strand elongation at 72~ for 1 min, and final elongation at 72~ for 5 rain. Following PCR, 25/.d of reaction were digested with 10 units of the appropriate restriction enzyme and then electrophoresed in a 4% agarose gel. Microsatellite polymorphic loci CI3-946, CI3-830, CI3-771, CI3-373, and GLUT2 were tested for LOH according to Jones and Nakamura (9). In detail, PCRs were carried out in a 10-/~1 reaction volume with 10 pmol of each primer; 50 ng of genomic DNA; 50mM KC1, 1.5mM MgCla, 10mM Tris-HC1 pH 8.3, 1% gelatin (w/v); 200 ~M concentrations each of dATP, dGTP, and dTFP; 50 /zM dCTP; 0.3 p.Ci of [a-32p]dCTP (3000 Ci/mmol; Amersham, Aylesbury, United Kingdom); and 0.25 unit of Taq DNA polymerase (Perkin Elmer Cetus). Reactions were performed as described above and consisted of 35 cycles. Following PCR, 2/~1 of the reaction were heat-denaturated and electrophoresed through a 6% polyacrylamide/7 M urea sequencing gel. Samples were considered informative for CA-dinucleotide repeat microsatellite polymorphisms for alleles separated by more than 4 base pairs. Ascertainment of LOH was performed by densitometric analysis comparing the ratio between the signal intensity of the two alleles in the neoplastic DNA with the same ratio in the corresponding normal DNA. LOH was defined as reduction in the allelic ratio >50%.