title: Identification and functional validation of HPV-mediated hypermethylation in head and neck squamous cell carcinoma.
abstract: BACKGROUND
  Human papillomavirus-positive (HPV+) head and neck squamous cell carcinoma (HNSCC) represents a distinct clinical and epidemiological condition compared with HPV-negative (HPV-) HNSCC. To test the possible involvement of epigenetic modulation by HPV in HNSCC, we conducted a genome-wide DNA-methylation analysis.
  
  
  METHODS
  Using laser-capture microdissection of 42 formalin-fixed paraffin wax-embedded (FFPE) HNSCCs, we generated DNA-methylation profiles of 18 HPV+ and 14 HPV- samples, using Infinium 450 k BeadArray technology. Methylation data were validated in two sets of independent HPV+/HPV- HNSCC samples (fresh-frozen samples and cell lines) using two independent methods (Infinium 450 k and whole-genome methylated DNA immunoprecipitation sequencing (MeDIP-seq)). For the functional analysis, an HPV- HNSCC cell line was transduced with lentiviral constructs containing the two HPV oncogenes (E6 and E7), and effects on methylation were assayed using the Infinium 450 k technology.
  
  
  CONCLUSIONS
  Unsupervised clustering over the methylation variable positions (MVPs) with greatest variation showed that samples segregated in accordance with HPV status, but also that HPV+ tumors are heterogeneous. MVPs were significantly enriched at transcriptional start sites, leading to the identification of a candidate CpG island methylator phenotype in a sub-group of the HPV+ tumors. Supervised analysis identified a strong preponderance (87%) of MVPs towards hypermethylation in HPV+ HNSCC. Meta-analysis of our HNSCC and publicly available methylation data in cervical and lung cancers confirmed the observed DNA-methylation signature to be HPV-specific and tissue-independent. Grouping of MVPs into functionally more significant differentially methylated regions identified 43 hypermethylated promoter DMRs, including for three cadherins of the Polycomb group target genes. 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 seen in HPV+ HNSCC tumors, and established E6 as the main viral effector gene.
  
  
  CONCLUSIONS
  Our data establish that archival FFPE tissue is very suitable for this type of methylome 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 tumor progression and metastasis.
authors: UCL Cancer Institute, University College London, 72 Huntley Street, London, WC1E 6DD ; Head and Neck Centre, University College London Hospitals NHS Trust, Euston Road, London, NW1 2PG.
authors: Matthias
authors: M
authors: Lechner
authors: UCL Cancer Institute, University College London, 72 Huntley Street, London, WC1E 6DD.
authors: Tim
authors: T
authors: Fenton
authors: UCL Cancer Institute, University College London, 72 Huntley Street, London, WC1E 6DD.
authors: James
authors: J
authors: West
authors: UCL Cancer Institute, University College London, 72 Huntley Street, London, WC1E 6DD.
authors: Gareth
authors: G
authors: Wilson
authors: UCL Cancer Institute, University College London, 72 Huntley Street, London, WC1E 6DD.
authors: Andrew
authors: A
authors: Feber
authors: UCL Cancer Institute, University College London, 72 Huntley Street, London, WC1E 6DD.
authors: Stephen
authors: S
authors: Henderson
authors: UCL Cancer Institute, University College London, 72 Huntley Street, London, WC1E 6DD.
authors: Christina
authors: C
authors: Thirlwell
authors: UCL Cancer Institute, University College London, 72 Huntley Street, London, WC1E 6DD.
authors: Harpreet K
authors: HK
authors: Dibra
authors: Department of Histopathology, University College London Hospitals NHS Trust, Rockefeller Building, University Street, London, WC1E 6JJ.
authors: Amrita
authors: A
authors: Jay
authors: UCL Cancer Institute, University College London, 72 Huntley Street, London, WC1E 6DD.
authors: Lee
authors: L
authors: Butcher
authors: UCL Cancer Institute, University College London, 72 Huntley Street, London, WC1E 6DD.
authors: Ankur R
authors: AR
authors: Chakravarthy
authors: UCL Cancer Institute, University College London, 72 Huntley Street, London, WC1E 6DD.
authors: Fiona
authors: F
authors: Gratrix
authors: UCL Cancer Institute, University College London, 72 Huntley Street, London, WC1E 6DD.
authors: Nirali
authors: N
authors: Patel
authors: Head and Neck Centre, University College London Hospitals NHS Trust, Euston Road, London, NW1 2PG.
authors: Francis
authors: F
authors: Vaz
authors: Head and Neck Centre, University College London Hospitals NHS Trust, Euston Road, London, NW1 2PG.
authors: Paul
authors: P
authors: O'Flynn
authors: Head and Neck Centre, University College London Hospitals NHS Trust, Euston Road, London, NW1 2PG.
authors: Nicholas
authors: N
authors: Kalavrezos
authors: UCL Cancer Institute, University College London, 72 Huntley Street, London, WC1E 6DD.
authors: Andrew E
authors: AE
authors: Teschendorff
authors: UCL Cancer Institute, University College London, 72 Huntley Street, London, WC1E 6DD.
authors: Chris
authors: C
authors: Boshoff
authors: UCL Cancer Institute, University College London, 72 Huntley Street, London, WC1E 6DD.
authors: Stephan
authors: S
authors: Beck