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Title: Oral squamous cell carcinomas and their genetic variants in association with extra capsular spread
Author: Bhattacharya, P.
Awarding Body: University of Liverpool
Current Institution: University of Liverpool
Date of Award: 2018
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Oral squamous cell carcinoma (OSCC) is the sixth most common cancer worldwide with an incidence of ~600,000 cases per year and younger age groups, particularly between 40-69 years, are increasingly becoming affected. OSCC has an overall five-year survival of only ~30% when metastatic disease is present. The most adverse prognostic clinical predictor for OSCC is extracapsular spread (ECS) from lymph node metastasis. The molecular determinants of ECS are undetermined and their characterization could significantly assist patient management, identifying those having increased risk and suggesting new possibilities for therapy. The aim of this study was to identify genetic variants associated with ECS. Previous work using aCGH had found an unexpectedly low frequency of TP53 alteration in OSCCs from a local cohort enriched for cases with ECS. SNP genotyping was therefore used to further investigate allelic imbalance of TP53 and for comparison CCND1 in primary tumour samples from the series (n=46; 19 node negative [N-], 7 [N+ECS-], 20 [N+ECS-]). CCND1 gain and TP53 loss were found to be associated with nodal status but not ECS. Five OSCC primary tumour cell lines (2 N- & 3 N+ECS+) were then screened by whole genome sequencing for genetic variants that may be associated with ECS. Variants were found in a broad range of genes but poor coverage (< 5 fold mean coverage) from some regions of the genome, in particular NOTCH1, suggested that there may have been technical limitations. However, pathway analysis implicated the NOTCH pathway may have had significance, consistent with literature reports. Additional next generation sequencing assays targeting NOTCH1 were therefore developed and used to analyze the gene in these cell lines. High coverage (150-200 fold mean coverage) of NOTCH1 sequences were achieved and in one of the ECS+ve cell lines (Liv7K), a rare, potentially deleterious nonsynonymous polymorphism (rs61751543) was found. Further screening for NOTCH1 and TP53 genetic variants associated with ECS was therefore performed for primary tumour samples (n=50; 21[N-], 11 [N+ECS-], 18 [N+ECS-]). Sanger resequencing was used for independent confirmation of candidate variants identified. Poorly covered samples (n=10) were excluded. 7/40 (17.5%) samples showed NOTCH1 variants, with 6/14 (43%) ECS+ samples confirmed to have NOTCH1 variants, compared to 1/26 (3.8%) ECS-, Fisher’s exact test p =0.0044. None of these samples displayed variants in TP53. Using this approach, comparing the frequency of the variants to node status, we produced evidence for a unique subset of ECS positive OSCC cases characterized by having disruption of NOTCH1 and absence of TP53 alteration. The NOTCH1 variants were clustered within the region for the extracellular domain (ECD), suggesting that intercellular communication and response could be disrupted. Further functional studies using an ECS +ve primary OSCC cell line as well as invasive and noninvasive controls, showed invasive cell lines demonstrated decreased cell growth and migration in response to Notch inhibition by DAPT (25 μM) treatment, with western blotting revealing absence of Notch1 and 3 after week three and Notch4 by week four only in Liv7K. These findings suggested the importance of further functional studies to assess the effect of Notch inhibition on markers of aggressive phenotype. Taken together our observations overall pave the way to future studies exploring the downstream effects of NOTCH1 ECD mutations and their potential role in OSCC.
Supervisor: Sibson, David Ross Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral