Oral squamous cell carcinoma (SCC) is the 6th most common malignancy in the US and is often associated with a history of tobacco and alcohol use. Five year survival and prognosis remain poor at 60-80% with the outcome becoming bleaker upon metastasis to the cervical (neck) lymph nodes (25-40%). Oral SCCs are preceded by oral dysplasias (precancers), but not all oral dysplasias turn malignant. Thus, challenges facing oral cancer surgeons and oncologists include prediction of which dysplasias will undergo malignant transformation and which tumors will metastasize to the neck. Therefore there is an urgent need to identify accurate biomarkers to aid in the treatment and management of patients with oral cancers or pre-cancers. This thesis research addressed these problems by assessing whether genomic DNA copy number measurements could provide such markers. As described below, the work has culminated in the identification of two oral SCC subtypes that differ in clinical behavior, specifically the risk of metastasis. The genomic copy number biomarker is suitable for stratifying patients for risk of metastasis prior to surgery and so can guide surgical planning for one of the most challenging treatment decisions - how to treat patients with clinically node-negative (N0) necks. Current clinical practice recommends removal of the cervical lymph nodes at the time of surgery to resect the tumor, because salvage of patients who subsequently develop lymph node metastasis has a poor success rate. Identification of patients with low risk for metastasis would spare them additional major surgery with its risks and morbidity, as well as reduce medical costs. A molecular marker for metastasis risk will also inform follow up management of oral cancer patients.

Studies of the Carcinoma-Associated Fibroblast in Human Oral Squamous Cell Carcinoma

S. Thaddeus Connelly

Abstract

Our understanding of the development and progression of oral squamous cell carcinoma (Oral SCC) remains limited. This is reflected by the fact that oral cancer is associated with one of the poorest 5-year survival rates of any malignancy in the body. It is becoming increasingly evident that the underlying stroma has an important role to play in tumorogenesis. Our studies focus on the central controller of the stroma, the fibroblast and its tumor-associated counterpart, the carcinoma-associated fibroblast (CAF). The molecular and functional characteristics of CAFs associated with Oral SCC remain incomplete. Thus, we studied the influence of the stroma on the tumor microenvironment through a histologic evaluation of cancerous and pre-cancerous lesions. Additionally, we assessed the molecular response that CAFs and fibroblasts display to TGFβ or TGFβ-mediated stimulation by direct measurement of expression levels of downstream genes and in an organotypic co-culture system, respectively. Lastly, we used expression profiling, immunofluorescence and functional assays of CAFs and normal fibroblasts (NFs) in an attempt to classify a CAF specific expression signature, CAF subsets or cellular origin and to identify CAF genes or gene pathways that might contribute to proliferation, migration and invasion. The results demonstrate that the stromal lymphocytic infiltrate increases with lesion severity of pre-cancers with the most in Oral SCC, indicating a causal relationship. Fibroblast response to TGFβ or TGFβ-mediated stimulation was varied based on anatomic site and this heterogeneity was further appreciated in the results of the immunofluorescence and expression profiling, however these studies confirmed that αSMA is a robust CAF marker. Despite the heterogeneity in the CAF/NF expression profiles, a set of genes was found and validated to be differentially expressed between gingiva CAFs and NFs and tongue and gingival fibroblasts in general. Finally, analysis of fibroblast groups defined through functional assays reveal differential gene expression between fibroblasts that are most or least inhibitory to keratinocyte/tumor cell line proliferation and those that increase or decrease invasion/migration. SFRP2 from the proliferation assay and DKK1 from the invasion/migration assay are both differentially expressed negative modulators of WNT signaling, indicating a potentially important pathway in the reciprocal tumor/stroma evolutionary relationship.

Background: Genomic DNA copy number aberrations are frequent in solid tumors, although the underlying causes of chromosomal instability in tumors remain obscure. Genes likely to have genomic instability phenotypes when mutated ( e. g. those involved in mitosis, replication, repair, and telomeres) are rarely mutated in chromosomally unstable sporadic tumors, even though such mutations are associated with some heritable cancer prone syndromes. Methods: We applied array comparative genomic hybridization (CGH) to the analysis of breast tumors. The variation in the levels of genomic instability amongst tumors prompted us to investigate whether alterations in processes/genes involved in maintenance and/or manipulation of the genome were associated with particular types of genomic instability. Results: We discriminated three breast tumor subtypes based on genomic DNA copy number alterations. The subtypes varied with respect to level of genomic instability. We find that shorter telomeres and altered telomere related gene expression are associated with amplification, implicating telomere attrition as a promoter of this type of aberration in breast cancer. On the other hand, the numbers of chromosomal alterations, particularly low level changes, are associated with altered expression of genes in other functional classes (mitosis, cell cycle, DNA replication and repair). Further, although loss of function instability phenotypes have been demonstrated for many of the genes in model systems, we observed enhanced expression of most genes in tumors, indicating that over expression, rather than deficiency underlies instability. Conclusion: Many of the genes associated with higher frequency of copy number aberrations are direct targets of E2F, supporting the hypothesis that deregulation of the Rb pathway is a major contributor to chromosomal instability in breast tumors. These observations are consistent with failure to find mutations in sporadic tumors in genes that have roles in maintenance or manipulation of the genome.