Genetic heterogeneity in saliva from patients with oral squamous carcinomas: implications in molecular diagnosis and screening.

We performed microsatellite analysis at chromosomal regions frequently altered in head and neck squamous carcinoma on matched saliva and tumor samples from 37 patients who had oral squamous carcinoma. The results were correlated with the cytologic findings and traditional clinicopathologic factors to assess the diagnostic and biological potential of these markers. Our data showed that 18 (49%) of the saliva samples and 32 (86%) of the tumors had loss of heterozygosity (LOH) in at least one of the 25 markers studied. In saliva, the combination of markers D3S1234, D9S156, and D17S799 identified 13 (72.2%) of the 18 patients with LOH in saliva (P < 0.001). For tumors, markers D3S1234, D8S254, and D9S171 together identified 27 (84.3%) of the 32 tumors with LOH at any of the loci tested (P < 0.001). Eleven (55%) of the 20 saliva samples with cytologic atypia and seven (35%) of the 17 specimens without atypia had LOH. Significant correlation between LOH in tumor at certain markers and smoking and alcohol use was found. Our results indicate that: 1) epithelial cells in saliva from patients with head and neck squamous tumorigenesis provide suitable material for genetic analysis; 2) combined application of certain markers improves the detection of genetic alteration in these patients; 3) clonal heterogeneity between saliva and matching tumor supports genetic instability of the mucosal field in some of these patients; and 4) LOH at certain chromosomal loci appears to be associated with smoking and alcohol consumption.

Molecular genetic alterations in carcinoma ex-pleomorphic adenoma: a putative progression model?

To determine the genetic changes associated with the development of carcinoma ex-pleomorphic adenoma (Ca Ex-PA), we analyzed 15 microsatellite loci at chromosome arms 8q, 12q, and 17p on DNA from 26 neoplasms (including 8 microdissected benign and malignant components), and 13 pleomorphic adenomas for comparison. Pleomorphic adenomas and the adenoma component of Ca Ex-PAs showed a higher incidence of loss of heterozygosity (LOH) at chromosome arms 8q (52%) and 12q (28%) than at 17p (14%) loci. In the carcinoma component, the combined LOH at chromosome arm 8q, 12q, and 17p regions was 69%, 50%, and 69%, respectively; within these chromosomal regions, 8q11.23-q12 (42%), 12q23-qter (39%), 17p13 (41%), and 17p11 (45%) loci manifested the highest incidence of LOH. Eight carcinomas (30.7%) showed loss at all three chromosomal arms tested. Of the eight microdissected Ca Ex-PAs analyzed, four adenoma and corresponding carcinoma components (50%) had the same LOH at 12q loci and additional LOH at 17p loci only in carcinomas. Chromosome arm 17p alterations correlated significantly with high disease stage and an increased proliferative rate in these tumors. Our results indicate that alterations at regions on chromosome arms 8q and/or 12q may constitute early events associated with pleomorphic adenomas; that LOH at 12q loci may identify a subset of adenoma with potential progression to carcinoma; that acquisition of additional alterations at chromosome arm 17p loci might represent an event preceding malignant transformation and progression; and that 8q, 12q, and 17p regions may harbor tumor suppressor genes involved in the genesis of PA and Ca Ex-PA. Genes Chromosomes Cancer 27:162-168, 2000.

Localization of chromosome 8p regions involved in early tumorigenesis of oral and laryngeal squamous carcinoma.

We analysed 30 primary invasive oral and laryngeal squamous carcinomas (SC), with concurrent dysplastic lesions, for genetic alterations at 15 microsatellite loci on the short arm of chromosome 8. Overall, loss of heterozygosity (LOH) was observed, in at least one informative locus, in 27% of the dysplastic lesions and in 67% of the invasive carcinomas. The highest frequency of allele losses in dysplasia (20% and 17%), and invasive carcinoma (40% and 48%) were detected in the same D8S298 and LPL-tet loci located on chromosomes 8p21 and 8p22 respectively. The minimal region with LOH was limited to 4.6 megaBases (mBs) at 8p22 and 7.1 mBs at 8p21. In addition, allelic losses in both dysplastic and corresponding invasive specimens were noted at the same loci in some tumors suggesting their emergence from a common preneoplastic clone. Allele losses correlated significantly with male gender, oral and laryngeal sites and high proliferative index. The data suggest that inactivation of tumor suppressor gene(s), within these loci, may constitute an early event in the evolution of oral and laryngeal SC.

Genotypic analysis of flow-sorted and microdissected head and neck squamous lesions by whole-genome amplification.

To investigate the utility of primer extension preamplification (PEP) in the genetic analysis of head and neck squamous tumorigenesis, microsatellite analysis was performed on matched deoxyribonucleic acid (DNA) samples extracted from 32 flow-sorted and microdissected specimens before and after PEP. Eighteen fresh and nine archival specimens were taken from invasive carcinomas, and five specimens were obtained from microdissected archival premalignant squamous epithelial lesions. Identical microsatellite patterns were observed in 276 (87%) of the 319 paired PEP and non-PEP genotypes with sufficient DNA. Overall, 13 (4%) of the PEP and 28 (8.8%) of the non-PEP fresh tissue samples failed specific microsatellite amplification. All 14 PEP-archival specimens were successfully amplified. Sorted cells showed a higher incidence (42.8%) of loss of heterozygosity (LOH) in both PEP and non-PEP samples compared with their unsorted counterparts. The results of this study indicate that (a) PEP is a simple and reliable technique for enhancing the DNA yield from small specimens; (b) flow sorting, in certain cases, improves the interpretation of genetic results; and (c) PEP may be used to compensate for PCR failure of unamplified DNA specimens in these lesions.