Molecular classification of HPV16-associated anal cancers: prognosis is dependent on major transcriptional regulation of E2F responsive genes Free

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Human papillomavirus (HPV) is a major etiological agent for the large majority of anal carcinomas. However, little is known as how HPV virus infection affects global gene expression patterns in anal carcinomas and how this influence is related to patient outcome. We here describe an analysis of the relation between HPV16-infection and gene expression patterns in a collection of anal cancer biopsies.

Global gene expression was analyzed by ABI microarray technology in 13 anal carcinoma biopsies positive for HPV subtype 16, and 4 biopsies of normal anal-mucosa. Quantitative real-time PCR analysis verified a subset of the resulting expression profiles.
 >Gene expression profiling and unsupervised hierarchical clustering divided the tumor biopsies in two groups with major differences in gene expression patterens; one (Gr1) closely related, the other (Gr2) more distantly related to normal tissue (NT).
 >Expression of HPV16 E7 mRNA was more elevated in Gr2. We show that the distinction into two groups could be impersonated by isolating expression profiles of known E2F regulated genes or genes induced specifically by high-risk HPV E6 and E7, and was accompanied by elevated mRNA level of HPV16 E7.
 >Statistical analysis between the two tumor groups revealed several differentially regulated key biological processes, involving genes involved in DNA replication, key regulators of the cell cycle, cell-cell adhesion or desmosome formation, transcriptional regulators, structural proteins and apoptosis.
 >To further investigate the relation between gene expression and prognosis, we analyzed the protein expression of mcm7, an e2f regulated gene typically representing the DNA replication cluster found in Gr2. The level of MCM7 protein expression was analyzed by tissuemicroarray and immunohistochemistry in an independent cohort of 55 anal cancer patients, treated with radiation and chemotheraphy.We found, that in an independent collection of anal cancer biopsies, high protein expression of the E2F regulated protein MCM7 was associated with favorable prognosis, both concerning disease specific survival and overall survival. Thus, massive influence on gene expression by high-risk HPV is associated with favorable prognosis, but this influence cannot be explained by the presence of HPV-16 virus alone.