Methylation status of HPV16 E2-binding sites classifies subtypes of HPV-associated oropharyngeal cancers.
BACKGROUND
The human papillomavirus (HPV) E2 protein is a transcriptional repressor of the oncogenes E6/E7 and loss of E2 function is considered a key step in carcinogenesis. Integration of HPV into the host genome may disrupt the E2 gene. Furthermore, methylation of CpG dinucleotides in E2-binding sites (E2BSs) in the HPV upstream regulatory region may interfere with transcriptional repression of E6 and E7 by E2. The authors hypothesized that the CpG methylation status of E2BS identifies subtypes of HPV type 16 (HPV16)-associated oropharyngeal squamous cell cancers (OPSCC) in association with E2 gene integrity and viral integration.


METHODS
Methylation of 10 CpG dinucleotides within the upstream regulatory region, encompassing E2BSs 1, 2, 3, and 4, was quantitatively analyzed by bisulfite pyrosequencing in 57 HPV16-associated OPSCC cases. E2 status was analyzed by gene amplification and quantitative real-time reverse transcriptase-polymerase chain reaction. Viral integration was determined by integration-specific polymerase chain reaction methods.


RESULTS
Three subgroups with differential methylation at E2BS3 and E2BS 4 were identified: 1) complete methylation (>80%) associated with the presence of integrated HPV genomes with an intact E2 gene; 2) intermediate methylation levels (20%-80%) with predominantly episomal HPV genomes with intact E2; and 3) no methylation (<20%) with a disrupted E2 gene. Patients with high methylation levels tended to have a worse 5-year overall survival compared with patients with intermediate methylation (hazard ratio, 3.23; 95% confidence interval, 1.13-9.24 [P = .06]).


CONCLUSIONS
Methylation of E2BS3 and E2BS4 in OPSCC is associated with E2 integrity and viral physical status. It might explain deregulated viral oncogene expression in the presence of E2. The prognostic significance of E2BS methylation for patients with HPV-associated OPSCC needs to be analyzed further.
Department of Applied Tumor Biology, Institute of Pathology, University of Heidelberg, Heidelberg, Germany.|Clinical Cooperation Unit, German Cancer Research Center, Heidelberg, Germany.
Miriam
M
Reuschenbach
Jean-Uhrmacher Institute for Oto-Rhino-Laryngological Research, University of Cologne, Cologne, Germany.
Christian U
CU
Huebbers
Department of Applied Tumor Biology, Institute of Pathology, University of Heidelberg, Heidelberg, Germany.|Clinical Cooperation Unit, German Cancer Research Center, Heidelberg, Germany.
Elena-Sophie
ES
Prigge
Institute of Medical Biometry and Informatics, University of Heidelberg, Heidelberg, Germany.
Justo Lorenzo
JL
Bermejo
Department of Applied Tumor Biology, Institute of Pathology, University of Heidelberg, Heidelberg, Germany.|Clinical Cooperation Unit, German Cancer Research Center, Heidelberg, Germany.
Martin S
MS
Kalteis
Department of Otorhinolaryngology, Head and Neck Surgery, University Hospital of Cologne, Cologne, Germany.
Simon F
SF
Preuss
Department of Otorhinolaryngology, Head and Neck Surgery, University Hospital of Cologne, Cologne, Germany.
Inga M C
IM
Seuthe
Jean-Uhrmacher Institute for Oto-Rhino-Laryngological Research, University of Cologne, Cologne, Germany.
Jutta
J
Kolligs
Department of Pathology, Maastricht University Medical Center, Maastricht, The Netherlands.
Ernst-Jan M
EJ
Speel
Department of Otorhinolaryngology, Maastricht University Medical Center, Maastricht, The Netherlands.
Nadine
N
Olthof
Department of Otorhinolaryngology, Maastricht University Medical Center, Maastricht, The Netherlands.
Bernd
B
Kremer
Department of Otorhinolaryngology, Head and Neck Surgery, University of Giessen, Giessen, Germany.
Steffen
S
Wagner
Department of Otorhinolaryngology, Head and Neck Surgery, University of Giessen, Giessen, Germany.
Jens P
JP
Klussmann
Department of Applied Tumor Biology, Institute of Pathology, University of Heidelberg, Heidelberg, Germany.|Clinical Cooperation Unit, German Cancer Research Center, Heidelberg, Germany.
Svetlana
S
Vinokurova
Department of Applied Tumor Biology, Institute of Pathology, University of Heidelberg, Heidelberg, Germany.|Clinical Cooperation Unit, German Cancer Research Center, Heidelberg, Germany.
Magnus
M
von Knebel Doeberitz
D001665:Binding Sites; D045744:Cell Line, Tumor; D019175:DNA Methylation; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D005260:Female; D052162:Human papillomavirus 16 / Q000145:classification / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D008297:Male; D008875:Middle Aged; D009856:Oncogene Proteins, Viral / Q000235:genetics* / Q000378:metabolism; D009959:Oropharyngeal Neoplasms / Q000821:virology*; D027383:Papillomaviridae / Q000235:genetics; D030361:Papillomavirus Infections / Q000821:virology*
D004268:DNA-Binding Proteins; C054855:E2 protein, Human papillomavirus type 16; D009856:Oncogene Proteins, Viral
E2-binding sites; human papillomavirus (HPV); methylation; oropharyngeal cancer; upstream regulatory region