Lechner, M; (2012) Genomic and Epigenomic Analysis of HPV positive and HPV negative Head and Neck Squamous Cell Cancer. Doctoral thesis , UCL (University College London).

Preview

PDF Lechner_final_thesis_redacted.pdf Download (4MB)

Abstract

Human Papillomavirus positive (HPV+) head and neck squamous cell carcinoma (HNSCC) represents a distinct clinical and epidemiological entity compared with HPV negative (HPV-) HNSCC. In this thesis I conducted both an epigenomic and genomic analysis to test the possible involvement of epigenetic modulation by HPV in HNSCC and associated genetic changes. Using laser-capture microdissection of formalin-fixed paraffin-embedded (FFPE) HNSCCs, I generated both DNA methylation and genetic profiles of HPV+ and HPV— samples. I then used an independent clinical sample set and HPV+ and HPV- HNSCC cell lines for the validation of the obtained methylation data by two independent methods (Infinium 450k BeadArray and MeDIP-seq). Paired end sequencing of captured DNA, representing 3,230 exons in 182 genes often mutated in cancer was applied for mutation profiling. I validated the latter findings by Infinium copy number variation (CNV) profiling, Sequenom MassArray sequencing and immunohistochemistry. Significant differences in the methylation and genomic profiles between HPV+ and HPV- HNSCC were observed. Methylation analysis revealed a hypermethylation signature with involvement of Cadherins of Polycomb group target genes in HPV+ HNSCC samples. Integration with independent expression data showed strong negative correlation, especially for the Cadherin gene family members. Combinatorial ectopic expression of the two HPV oncogenes (E6 and E7) in an HPV— HNSCC cell line partially phenocopied the hypermethylation signature observed in HPV+ HNSCC tumours and established E6 as the main viral effector gene. Moreover, MeDIP-Seq data revealed methylation sites within integrated HPV genomes. These methylation sites were confirmed by bisulfite sequencing, both in HNSCC samples and HPV+ HNSCC cell lines. Validated genomic changes clustered HPV+ and HPV- oropharyngeal carcinomas into two distinct subgroups with TP53 mutations detected in 100% of HPV- cases. Abrogation of the G1/S checkpoint by CCND1 amplification and CDKN2A loss occurred in the majority of HPV- tumours, indicating that trials with CDK inhibitors in this disease subtype may be warranted. My data establish archival FFPE tissue to be highly suitable for these types of methylation and mutation analysis and suggest that HPV modulates the HNSCC epigenome through hypermethylation of Polycomb repressive complex 2 target genes such as Cadherins which are implicated in tumour progression and metastasis. Moreover, my findings reinforce the causal role of HPV in oropharyngeal cancer and indicate that therapeutic stratification according to somatic genomic changes, in addition to HPV status, could be the most appropriate future approach for these cancers.

Download activity - last month

Export as CSVJSONXML

Download activity - last 12 months

Export as CSVXMLJSON

Downloads by country - last 12 months

Export as XMLJSONCSV

Archive Staff Only

View Item