Use of complete coverage array comparative genomic hybridization to define copy number alterations on chromosome 3p in oral squamous cell carcinomas

Loss of 3p has been associated with oral cancer progression and is common in many cancers. However, regions of alteration on 3p are poorly defined. We have constructed a high-resolution chromosomal array using a tiling set of 535 human bacterial artificial chromosomes that provides near complete coverage of 3p. Array comparative genomic hybridization analysis of 20 microdissected oral squamous cell carcinomas showed multiple and recurrent segments of copy number changes. These include a deletion containing the FHIT gene; novel segments of copy decrease at 3p22, 3p24, and 3p26; and an unexpected approximately 0.7 Mbp segmental increase at 3p21. These data strongly support the value of using chromosomal array comparative genomic hybridization for detailed profiling of oral squamous cell carcinomas.     

Multiple microalterations detected at high frequency in oral cancer

The development of array comparative genomic hybridization (array CGH) at tiling-path resolution has enabled the detection of gene-sized segmental DNA copy number gains and losses. Here, we present the first application of whole genome tiling-path array CGH to archival clinical specimens for the detailed analysis of oral squamous cell carcinomas (OSCC). We describe the genomes of 20 OSCCs as well as a selection of matched normal DNA in unprecedented detail. Examination of their whole genome profiles enabled the identification of alterations ranging in size from whole-arm, segmental, to gene size alterations. Tiling-path resolution enabled the detection of many more alterations within each tumor than previously reported, many of which include narrow alterations found to be frequent events among the 20 OSCCs. We report the presence of several novel frequent submegabase alterations, such as the 0.58 Mb gain at 5p15.2 containing triple functional domain (TRIO), detected in 45% of cases. We also report the first coamplification of two gene clusters, by fine-mapping the precise base pair boundaries of the high-level amplification at 11q22.2-22.3 containing both matrix metalloproteinase and baculoviral IAP repeat-containing protein 2 (BIRC) gene clusters. These results show the large improvement in detection sensitivity and resolution compared with genome interval marker arrays and the utility of tiling resolution array CGH for the detection of both submegabase and single copy gains and losses in cancer gene discovery.     

Alteration of AKAP220, an upstream component of the Rb pathway, in oral carcinogenesis

Few genes have been implicated in the development of oral cancer. In our study, we identified a novel gene in the Rb pathway that is frequently altered and overexpressed in oral tumors. Significantly, the alteration is also associated with early oral premalignant lesions (OPLs). This region was identified through a genomewide scan using randomly amplified polymorphic DNA (RAPD) PCR of 40 microdissected oral squamous cell carcinomas (SCCs). Recurrent gain of a approximately 400 bp signal was observed in multiple patients. This gain was localized to 13q14.11, a region frequently altered in multiple cancer types. Through microsatellite analysis, a 1.9 Mbp minimal region of alteration (MRA) was defined between D13S263 and D13S1227. Allelic imbalance (AI) in the MRA was present in only 28% of low-grade dysplasia, but strikingly increased with progression to 64% in high-grade dysplasia, plateauing at 61% in tumors, thus implicating this alteration in the early stages of disease development. Of the 3 genes residing within the MRA, Receptor Activator of NK-kappa-B Ligand (RANKL) and Diacylglycerol Kinase (DGKH) showed no change in expression levels in tumors compared to normal tissue. In contrast, 12 of 16 tumors showed significant overexpression of A-Kinase Anchoring Protein 220 (AKAP220). Since AKAP220 plays a role in regulating the Rb pathway, its dysregulation may contribute significantly to alterations in cell cycle regulation that facilitate progression of OPLs.