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Title: Molecular indicators and tumour models of extracapsular spread in metastatic oral squamous cell carcinoma
Author: Dhanda, Jagtar
ISNI:       0000 0004 5368 8726
Awarding Body: University of Liverpool
Current Institution: University of Liverpool
Date of Award: 2014
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Introduction Extracapsular spread (ECS) is the single most adverse prognosticator for recurrence and death in metastatic oral squamous cell carcinoma (OSCC) and relapse is common in both primary and metastatic sites. It is rarely exploited for biological investigation with limited study of its molecular determinants and the absence of a tumour model. The absence of a biomarker also means exhaustive histological examination after surgery is required for diagnosis. ECS is a late manifestation of the metastatic cascade and would be expected to be intimately related to interactions between tumour cells and the microenvironment. The importance of the tumour microenvironment has been highlighted by the discovery of a promising new stromal biomarker, alpha smooth muscle actin (ASMA), which has shown even greater prognostic value than ECS. The aims of this study are to identify and validate biomarkers for ECS and to develop and utilise ECS+ve primary cell culture-derived cell lines in organotypic models to investigate epithelial and mesenchymal interactions. Methods 102 patients treated with primary surgery for OSCC were utilised for biomarker discovery and validation. Diagnostic accuracy of MRI scanning for ECS was determined and compared to a previously determined 8-gene expression signature from primary site tissue, which was validated using quantitative real-time polymerase chain reaction (qRT-PCR). SERPINE1 and ASMA expression was assessed for prognostic capability by immunohistochemistry (IHC) on a case-matched tissue microarray with separate analysis of the tumour centre and the advancing-front. The explant technique was used with cell-type specific media to isolate fibroblast and keratinocyte single-cell populations from 47 OSCC biopsy tissues. Metastatic status of the originating tumours was correlated with establishment of cell lines, cell phenotypes, proliferation index, invasion in organotypic cultures and drug sensitivities. Short tandem repeat profiling and microsatellite instability analysis were used to determine the tissue of origin. Results MRI (n=88) showed very poor sensitivity for the detection of ECS (7%), while the 8 gene signature had a sensitivity of 78%. The qRT-PCR findings poorly correlated with the microarray findings, but SERPINE 1 and HEXIM1 were selected for IHC as the best performing genes from prediction models using receiver operating characteristic curves (AUC 0.59 and 0.64 respectively). IHC indicated that both SERPINE1 and ASMA expression at the tumour-advancing front was highly significantly associated with nodal/ECS status (p < 0.001). Both separately, and in combination, SERPINE1 and ASMA were superior to MRI and the 8-gene expression signature for the detection of ECS (sensitivity; SERPINE1: 95%; ASMA: 82%; combination: 81%). ECS was associated with expression of either or both proteins in all cases and ASMA+/SERPINE1+ expression, in combination, was highly significantly associated with adverse outcomes (p < 0.001). A significant association was found between nodal status and both successful generation of finite keratinocyte cell lines (p=0.04) and keratinocyte invasion (p < 0.001), with histopathological similarities observed between organotypic models of invasion and the corresponding in-situ tumour advancing fronts. Two out of 13 cell lines grew for >10 passages and both were associated with ECS and distant metastasis (p < 0.001). Morphological differences were observed between fibroblasts cultured from ECS-ve and ECS+ve disease, with increased Vimentin and ASMA expression associated with ECS+ disease together with an enhanced capacity to induce and alter invasive phenotypes in organotypic models (p < 0.001). The primary cell culture derived cells showed greater sensitivity to cisplatin and docetaxel (p=0.003 and p=0.007 respectively) compared to secondary cell lines. An incremental reduction in growth rates (p=0.005) and sensitivity to cisplatin (p=0.02) was seen with advancing passage and both cisplatin and docetaxel reduced invasion in organotypic models, with the effects of docetaxel more pronounced (p < 0.001). Microsatellite instability analysis suggested that the primary cell culture derived fibroblasts were not derived from tumour cells by epithelial to mesenchymal transition. Discussion These findings suggest that a combination of ASMA and SERPINE1 IHC offers promise in the search for biomarkers that can be used to stratify therapeutic approaches in OSCC using biopsy samples that include the invading edge of the tumour. Successful generation of keratinocyte cell lines and invasion in organotypic models is related to the aggressiveness of the primary tumour that can also predict for the presence of ECS thus showing both sites are related. Both the IHC and cell culture findings suggest a putative role for the tumour microenvironment in ECS. Organotypic models using primary cell culture derived cells simulate the morphological characteristics of invasion in the primary tissue advancing front supporting their role as an in vitro tumour model and, most significantly, indicate that fibroblasts influence the invasive phenotype. These cell lines have greater sensitivity to anti-cancer agents and may offer opportunities for investigating new stromal-targeted therapies for ECS.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available