Identification of allelic loss on chromosomes 8q and 13q in oral premalignancies and tumors

Oral squamous cell carcinoma (SCC) is believed to progress through sequential stages of premalignancies to invasive cancer. Once cancer is formed, the prognosis is poor and the 5year survival rate is less than 50%. It is important to develop new strategies to improve diagnosis in early stages and to obtain information on the molecular mechanisms underlying carcinogenesis. It is now widely accepted that cancer development is underlined with the accumulation of changes to the critical control genes. Analysis of lesions at different stages of carcinogenesis may help to define the genetic changes associated with these stages during the development and progression of oral SCC. Microsatellite analysis of loss of heterozygosity (LOH) has been shown to be a powerful molecular approach to detect loss of tumor suppressor genes (TSGs) in many organs and systems, including oral cancer and premalignant lesions. The first objective of this thesis was to fine map one region of deletion at 13ql4.11 that was previously identified by RAPD (random amplified polymorphic DNA), in search of novel regions (minimal region of alteration, MRA) containing tumor genes. Two other MRAs have been identified by other people in the research group at 8q22 (D8S545 and D8S1830). The second objective was to investigate the temporal changes of the 3 new hotspots (one to be identified at 13ql4.11 by this study and 2 at 8q22, D8S545 and D8S1830) during oral carcinogenesis, and compare this data with known regions of losses at chromosomes 3p, 9p, and 17p. The latter represent frequent early changes in this disease. DNA microdissected from 58 dysplasias and 40 SCCs were studied by microsatellite analysis for LOH. ii For fine mapping at 13ql4.11, 3 primers were used: D13S1297 within 13ql4.11, D13S263 (1.6 Mbp centromeric to D13S1297) and D13S1227 (0.3 Mbp telomeric to D13S1297). Sixteen tumors showed LOH at D13S1297. A centromeric boundary at D13S263 was observed in 6 of the 16 tumors and a telomeric boundary at D13S1227 in 4 of the 16 tumors. Similar results were found in dysplasias. Thirteen dysplasias showed LOH at D13S1297. A centromeric boundary at D13S263 was observed in 7 of the 13 dysplasias and a telomeric boundary at D13S1227 in 6 of the 13 dysplasias. The results suggest presence of a 1.9 Mb MRA spanning between D13S263 and D13S1227. For temporal changes, LOH at D13S1297 (13ql4.11) was present in 23% low-grade dysplasia, increasing in frequency with progression to high-grade dysplasia (58%) then plateauing in tumors (57%). Such high frequency of LOH at D13S1297 during oral carcinogenesis again supports the presence of tumor gene(s) at the newly identified MRA at D13S1297. Using the above results, another graduate student in the research group has gone further with the experiment and demonstrated that AKAP220, a gene located within the 1.9 Mb MRA, was overexpressed in oral SCCs. Our research group is the first to associate AKAP220 with tumorigenesis. High frequency of LOH was also noted in the other two new hotspots at 8q22 during the multistage oral carcinogenesis. LOH at D8S1830 was noted in 22 of the 38 (58%) informative low-grade dysplasias, 4 of 12 (33%) high-grade dysplasias, and 13 of 31 (42%) SCCs. LOH at D8S545 was noted to increase with progression of the lesions: in 4 of the 22 (18%) informative low-grade dysplasias, 3 of 12 (25%) high-grade dysplasias, and 11 of 26 (42%) SCCs. ) In summary, this thesis has identified a new hotspot at 13ql4.11 (D13S1297) and LOH analysis of different stages of oral lesions supported the presence of the new hotspot. A novel gene, AKAP220 has been identified within this region subsequent to this thesis (and based on its results). In addition, high frequency of LOH was also noted for the other two new hotspots at 8q22 and further studies are needed to identify the tumor genes within the regions.