Ivy F.L. Tsui

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.

Multiple Aberrations of Chromosome 3p Detected in Oral Premalignant Lesions

The study of oral premalignant lesions (OPL) is crucial to the identification of initiating genetic events in oral cancer. However, these lesions are minute in size, making it a challenge to recover sufficient DNA from microdissected cells for comprehensive genomic analysis. As a step toward identifying genetic aberrations associated with oral cancer progression, we used tiling-path array comparative genomic hybridization to compare alterations on chromosome 3p for 71 OPLs against 23 oral squamous cell carcinomas. 3p was chosen because although it is frequently altered in oral cancers and has been associated with progression risk, its alteration status has only been evaluated at a small number of loci in OPLs. We identified six recurrent losses in this region that were shared between high-grade dysplasias and oral squamous cell carcinomas, including a 2.89-Mbp deletion spanning the FHIT gene (previously implicated in oral cancer progression). When the alteration status for these six regions was examined in 24 low-grade dysplasias with known progression outcome, we observed that they occurred at a significantly higher frequency in low-grade dysplasias that later progressed to later-stage disease (P < 0.003). Moreover, parallel analysis of all profiled tissues showed that the extent of overall genomic alteration at 3p increased with histologic stage. This first high-resolution analysis of chromosome arm 3p in OPLs represents a significant step toward predicting progression risk in early preinvasive disease and provides a keen example of how genomic instability escalates with progression to invasive cancer.

A Dynamic Oral Cancer Field: Unraveling the Underlying Biology and Its Clinical Implication

Oral cancer is a complex disease that is characterized by histologic and genetic heterogeneity. The evolution and progression of this disease is thought to result from the accumulation of alterations in molecular pathways. Although the oral cavity is accessible for routine screening of suspicious lesions, gene alterations are known to accrue in histologically normal tissues. Therefore, some cancer forerunners may remain undetected clinically or histologically. Recently emerging optical and molecular technologies have provided a powerful means for redefining the extent of the field of alteration. Often this means expanding upon regions detectable with standard white light approaches. In this report, we used a newly developed optical technique, direct fluorescence visualization, to define a contiguous field that extended beyond the margins of a clinically visible oral squamous cell carcinoma. Multiple biopsies were taken within this contiguous optically altered field. Genome alterations detected for each specimen were compared to define whether each lesion arose independently or as a consequence of a shared progenitor cell. Our results indicate that the field effect of oral cancer is extremely dynamic, with different genetic alterations present in different biopsies within a field. This case study also demonstrated that 2 genetically unrelated squamous cell carcinoma could be developed within 10 mm at the right lateral tongue of this patient. These findings provide evidence for the importance to implement optical technologies in defining surgical margins and support the use of whole genome technologies in the diagnosis of clonal versus independent lesions of the oral cavity, which may have implications on treatment strategies.

Integrative molecular characterization of head and neck cancer cell model genomes

Cell lines are invaluable model systems for the investigation of cancer. Knowledge of the molecular alterations that exist within cell models is required to define the mechanisms governing cellular phenotypes.

Recurring DNA copy number gain at chromosome 9p13 plays a role in the activation of multiple candidate oncogenes in progressing oral premalignant lesions

Genomic alteration at chromosome 9p has been previously reported as a frequent and critical event in oral premalignancy. While this alteration is typically reported as a loss driven by selection for CDKN2A deactivation (at 9p21.3), we detect a recurrent DNA copy number gain of ~2.49 Mbp at chromosome 9p13 in oral premalignant lesions (OPLs) that later progressed to invasive lesions. This recurrent alteration event has been validated using fluorescence in situ hybridization in an independent set of OPLs. Analysis of publicly available gene expression datasets aided in identifying three oncogene candidates that may have driven selection for DNA copy number increases in this region (VCP, DCTN3, and STOML2). We performed in vitro silencing and activation experiments for each of these genes in oral cancer cell lines and found that each gene is independently capable of upregulating proliferation and anchorage‐independent growth. We next analyzed the activity of each of these genes in biopsies of varying histological grades that were obtained from a diseased oral tissue field in a single patient, finding further molecular evidence of parallel activation of VCP, DCTN3, and STOML2 during progression from normal healthy tissue to invasive oral carcinoma. Our results support the conclusion that DNA gain at 9p13 is important to the earliest stages of oral tumorigenesis and that this alteration event likely contributes to the activation of multiple oncogene candidates capable of governing oral cancer phenotypes.

Abstract 3041: Focal amplification of chromosome 9p13 is associated with progression risk and activation of multiple oncogenes in oral precancers

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Oral squamous cell carcinoma, the most common form of head and neck cancer, has a dismal five year survival rate of ∼50%. This is largely due to the late stage of diagnosis and high rates of recurrence. A better understanding of the mechanisms driving early oral tumorigenesis is an essential first step towards increasing survival rates, which have not improved in decades. Specifically, by characterizing genetic alterations in early oral premalignant lesions (OPLs), we will better understand mechanisms of disease progression and discover targets for timely therapeutic intervention that could ultimately improve oral cancer outcomes. Methods: Whole genome DNA copy number profiling was undertaken on a rare panel of OPLs with extensive longitudinal data (including known progression status for invasive disease). Recurring alterations were characterized and gene lists were refined using independent tumor-associated gene expression datasets. The functional significance of remaining gene candidates was then assessed using lentiviral shRNA and expression vectors in cell model systems. Results: Analysis of genomic data from OPLs identified four focal recurrent alterations. Three of these alterations were associated with previously identified oncogenes: cMYC, EGFR, and KRAS. The fourth was a novel 2.04 Mbp amplification at chromosome 9p13. Based on our results and publicly available gene expression data, four candidate genes were found in this region: VCP, DCTN3, STOML2, and TLN1. Independent manipulation of each of these genes in cell models revealed changes in oncogenicity (based on changes in rate of proliferation and the degree of anchorage independence), suggesting a critical role in malignant processes associated with cancer progression. Conclusion: We have shown that a recurring, focal 9p13 amplicon in OPLs is associated with progression to invasive oral cancer. Further, our data indicate that this region spans multiple genes with oncogenic potential. Taken together, our results suggest that a single amplification event may dysregulate multiple genes that drive progression to invasive oral cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3041. doi:10.1158/1538-7445.AM2011-3041

Abstract 3007: Integrative analysis of histologically distinct oral lesions obtained from a single disease field uncovers stage-specific molecular alterations

Oral cancer (OC) is a leading cause of cancer death worldwide, with late stage diagnosis and high recurrence rates accounting for poor survival statistics. Like many solid tumors, OC develops through a series of histopathological stages: from hyperplasia to dysplasia to carcinoma in situ (CIS) and finally to invasive cancer. While dysplastic lesions can be readily detected in clinic, they are rarely treated as only a small fraction will ultimately progress to invasive disease. By identifying the molecular mechanisms driving early malignant disease, novel tools can be developed for discriminating progression risk in early lesions and identify druggable targets that could greatly improve currently stagnant oral cancer survival rates. Objective: To identify genes critical to early oral tumorigenesis that may also have utility as biomarkers for disease outcomes. Methods: Fluorescence visualization techniques were used to define OC disease fields. For each patient multiple biopsies revealing distinct disease stages were simultaneously collected from within the disease field. DNA copy number (SMRT CGH), methylation (Illumina Infinium), and gene expression (Agilent 4×44K) profiles on all collected premalignant biopsies from each patient (hyperplasia, dysplasia, and CIS) were generated. Gene candidates were identified from integrated datasets based on current dogma. For example, genes exhibiting decreased expression and concurrent increased methylation, and vice versa, were flagged for further analysis. Recurring genetic and signaling cascade events were defined using Ingenuity Pathway Analysis software and validated by sequencing and expression analysis. Results: While no DNA copy number changes were observed in dysplastic biopsies, several changes were observed in DNA methylation and gene expression data. This suggests that methylation changes may play a critical role in gene deregulation for early oral lesions. As expected, methylation and expression alterations were observed to increase with disease severity. Several genes were found to be deregulated at a frequency of >50% in dysplastic lesions, strongly suggesting a critical role for these candidates in early disease development. Additional genes were recurrently altered during progression to CIS. Pathway analysis of recurrent genes candidates revealed a potential role of the wnt/β-catenin pathway and inflammation/immune response pathways in oral lesion development. Conclusions: By leveraging high throughput platforms and a rare collection of patient-matched oral precancer tissues, we have shown that aberrant methylation is a critical mechanism driving molecular dysregulation in early oral tumorigenesis. Specific candidates identified by our integrative approach may have utility as biomarkers or therapeutic targets, ultimately helping to improve oral cancer outcomes. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3007. doi:10.1158/1538-7445.AM2011-3007

Abstract C53: The genetic evolution of oral cancer fields

Introduction: The evolution of oral cancer results from the accumulation of genetic alterations. Field cancerization, where histologically or molecularly abnormal cells surround a clinically visible tumor to a wider extent, imposes a challenge to delineate surgical boundaries. We applied a newly emerging optical technique using direct fluorescence visualization (FV) to redefine the field of alteration. Using genomic profiling we examined multiple biopsies within the field to assess clonal expansion of cells within this optically altered field. Experimental Approach: A hand‐held FV device was used in the operating room to define the field that extended beyond the margins of clinically visible oral cancer. Multiple biopsies (N=15) were taken within the altered FV field (FV loss or FVL) and the surgical margins with no FVL, as controls, from three patients. Histological assessment and microdissection were performed for each biopsied sample. The genomic profile of each sample was generated using a tiling‐path DNA microarray. A breakpoint detection algorithm was used to define genetic breakpoints and clonal ordering was performed to infer the sequence of genetic events of samples within each patient. Result: Early stage low‐grade dysplasias were found within the FVL field in all patients, while no dysplasia was detected in the areas with no FVL. In general, each field is histologically and genetically heterogeneous. Specifically, patient A presented with a clinically identifiable SCC (#1), while another SCC (#3) was found in an area 10‐mm anterior to SCC#1, which was not clinically apparent but showed FVL. A moderate dysplasia (#2) was found between SCC#1 and SCC#3. Interestingly, 5q, 8p, and 8q loss were common among all three samples (suggesting a common progenitor), while genetic alterations (e.g., high‐level amplification on 9p22.3‐pter) accumulated in both SCC#3 and dysplasia#2 but was absent in SCC#1. On the other hand, SCC#1 accumulated different genetic changes (e.g., gain of 11q13.2‐q13.4 (CCND1)). This suggested that two clonal lineages were present within this cancerous field. Similarly, in patient B, the biopsies obtained revealed both common and different genetic signatures. For example, a moderate dysplasia showed genetic alterations specific to this lesion (e.g., high‐level amplification of 8q11.21 (SNAI2)), while its corresponding carcinoma in situ harbored numerous different genetic alterations, including three regions of high‐level amplification (e.g., 20q11.23 (SRC)). Genomic profiles of these samples suggest that two different genetic pathways diverged from a common progenitor, while subsequent genetic alterations accumulated for the formation of each unique subpopulation. In patient C, one genetic pathway was found governing the development of the clinically identifiable SCC, and increased genetic alterations were detected in the SCC compared to the mild dysplasia. All the controls did not show matching genetic changes. Conclusion: Our results indicate that the genetics of the oral cancer field is extremely dynamic, where different clones are evolving in the field. Genetic alterations occurring early in the genetic pathway may be important events that prime the area for further development of cancer. These findings provide evidence for the importance of implementing optical technologies in defining surgical margins as well as the importance of tailored targeted therapies to effectively treat different subclones of a field. Citation Information: Cancer Res 2009;69(23 Suppl):C53.