Multiple pathways in the FGF signaling network are frequently deregulated by gene amplification in oral dysplasias

Genetic alteration in oral premalignant lesions (OPLs), the precursors of oral squamous cell carcinomas (OSCCs), may represent key changes in disease initiation and development. We ask if DNA amplification occurs at this early stage of cancer development and which oncogenic pathways are disrupted in OPLs. Here, we evaluated 50 high‐grade dysplasias and low‐grade dysplasias that later progressed to cancer for gene dosage aberrations using tiling‐path DNA microarrays. Early occurrences of DNA amplification and homozygous deletion were frequently detected, with 40% (20/50) of these early lesions exhibiting such features. Expression for 88 genes in 7 recurrent amplicons were evaluated in 5 independent head and neck cancer datasets, with 40 candidates found to be overexpressed relative to normal tissues. These genes were significantly enriched in the canonical ERK/MAPK, FGF, p53, PTEN and PI3K/AKT signaling pathways (p = 8.95 × 10^?3 to 3.18 × 10^?2). These identified pathways share interactions in one signaling network, and amplification‐mediated deregulation of this network was found in 30.0% of these preinvasive lesions. No such alterations were found in 14 low‐grade dysplasias that did not progress, whereas 43.5% (10/23) of OSCCs were found to have altered genes within the pathways with DNA amplification. Multitarget FISH showed that amplification of EGFR and CCND1 can coexist in single cells of an oral dysplasia, suggesting the dependence on multiple oncogenes for OPL progression. Taken together, these findings identify a critical biological network that is frequently disrupted in high‐risk OPLs, with different specific genes disrupted in different individuals. © 2009 UICC     

Integrating the multiple dimensions of genomic and epigenomic landscapes of cancer

Advances in high-throughput, genome-wide profiling technologies have allowed for an unprecedented view of the cancer genome landscape. Specifically, high-density microarrays and sequencing-based strategies have been widely utilized to identify genetic (such as gene dosage, allelic status, and mutations in gene sequence) and epigenetic (such as DNA methylation, histone modification, and microRNA) aberrations in cancer. Although the application of these profiling technologies in unidimensional analyses has been instrumental in cancer gene discovery, genes affected by low-frequency events are often overlooked. The integrative approach of analyzing parallel dimensions has enabled the identification of (a) genes that are often disrupted by multiple mechanisms but at low frequencies by any one mechanism and (b) pathways that are often disrupted at multiple components but at low frequencies at individual components. These benefits of using an integrative approach illustrate the concept that the whole is greater than the sum of its parts. As efforts have now turned toward parallel and integrative multidimensional approaches for studying the cancer genome landscape in hopes of obtaining a more insightful understanding of the key genes and pathways driving cancer cells, this review describes key findings disseminating from such high-throughput, integrative analyses, including contributions to our understanding of causative genetic events in cancer cell biology.     

Novel regions of amplification on 8q distinct from the MYC locus and frequently altered in oral dysplasia and cancer

Genetic studies aimed at identifying key alterations in oral cancers have focused on analysis of tumors, with few such studies using early oral premalignant lesions (OPLs) because of limitations in both sample availability and size. In this study, we used a randomly amplified polymorphic DNA (RAPD)-PCR approach to fingerprint DNA from microdissected normal and dysplastic cells and identified two recurrent genetic alterations on the long arm of chromosome 8 in OPLs, one mapping to 8q22 and the other to 8q24 near the MYC locus. We constructed a high-resolution bacterial artificial chromosome (BAC) comparative genomic hybridization array consisting of 166 overlapping BAC clones that spans about 52 Mbp, from 8q21 to 8q24. Hybridization of DNA from microdissected oral tumors to the array revealed alteration at 8q24, with amplification of the BAC containing MYC. Strikingly, at least two other novel regions of amplification at 8q22 were identified. Microsatellite analysis of 93 oral dysplasias and tumors confirmed the presence of one of the alterations at 8q22. Loss of heterozygosity (LOH) at D8S1830, mapping within one of the regions of amplification, was observed in high frequency in both OPLs and tumors. Of the 37 cases with LOH at D8S1830, 23 (62%) showed retention at D8S1793, which maps 1.6 Mbp centromeric to MYC. This is further support for the alteration at 8q22 being distinct from MYC. These data raise the possibility of additional oncogenes on 8q near the MYC locus that are potentially involved in OPL disease progression.     

An efficient method for the assessment of DNA quality of archival microdissected specimens.

There will be an increasing need of methods for assessing the suitability of specimens for genetic-based assays as DNA markers become an integral part of molecular diagnosis. The targeting of specimens for specific analyses will require the ability to rapidly screen for DNA quality. Conventional methods such as Southern analysis and gene specific-polymerase chain reaction (PCR) often require quantities of material that represent a significant portion of the specimen, especially in microdissected samples. Here we describe a novel application of a commonly used PCR-based DNA-fingerprinting technology that requires minimal quantities of DNA to simultaneously assess multiple regions throughout the genome for DNA quality. Randomly amplified polymorphic DNA (RAPD) PCR generates DNA fragments of a broad size range with the product size reflecting the degree of sample fragmentation. Fourteen DNA samples extracted from cells microdissected from seven formalin-fixed, paraffin-embedded oral cancer biopsies were assessed for DNA quality using gene-specific PCR and RAPD-PCR. Although the more conventional assay required 2-ng DNA (or 300-cell equivalents) to examine DNA quality at a single locus, RAPD-PCR provided a more informative profile of DNA quality from the same microdissected archival specimens.     

Overexpression of LRP12, a gene contained within an 8q22 amplicon identified by high-resolution array CGH analysis of oral squamous cell carcinomas

Chromosome 8q amplification is a common event observed in cancer. In this study, we used high-resolution array comparative genomic hybridization to resolve two neighboring regions on 8q that are both amplified in oral cancer. One region (at 8q24) contains the MYC oncogene, which is frequently overexpressed in many cancers, while the other region (at 8q22) represents a novel amplicon. The alignment of array comparative genomic hybridization profiles of 20 microdissected oral squamous cell carcinomas (OSCCs) revealed a approximately 5 Mbp region of frequent copy number alteration. This region harbors 16 known genes. Gene expression analysis comparing 15 microdissected OSCC with 16 normal epithelium samples revealed overexpression specific to LRP12 but not the neighboring genes, dihydropyrimidinase and FOG2, suggesting that LRP12 may function as an oncogene in oral tumors.     

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.