TY - JOUR
T1 - CpG methylation analysis of hpv16 in laser capture microdissected archival tissue and whole tissue sections from high grade anal squamous intraepithelial lesions
T2 - a potential disease biomarker
AU - Molano, Monica
AU - Tabrizi, Sepehr N.
AU - Garland, Suzanne M.
AU - Roberts, Jennifer M.
AU - MacHalek, Dorothy A.
AU - Phillips, Samuel
AU - Chandler, David
AU - Hillman, Richard J.
AU - Grulich, Andrew E.
AU - Jin, Fengyi
AU - Poynten, I. Mary
AU - Templeton, David J.
AU - Cornall, Alyssa M.
AU - SPANC Study Team
AU - McCaffery, Kirsten
AU - Howard, Kirsten
AU - Farnsworth, Annabelle
AU - Fairley, Kit
AU - Prestage, Garrett
AU - Carr, Andrew
AU - Tong, Winnie
AU - Law, Carmella
AU - Acraman, Brian
AU - Crampton, Leonie
AU - Feeney, Lance
AU - Fraissard, Eddie
AU - Law, Matthew
AU - McGrath, Patrick
AU - Mellor, Robert
AU - Norris, Richard
AU - O'Dwyer, Matthew
AU - Pendlebury, Susan
AU - Petoumenos, Kathy
AU - Richards, Adele
AU - Schema, Lance
AU - Seeds, Daniel
AU - Segelov, Eva
AU - Thurloe, Julia
AU - Varma, Rick
N1 - Copyright the Author(s) 2016. Version archived for private and non-commercial use with the permission of the author/s and according to publisher conditions. For further rights please contact the publisher.
PY - 2016/8/16
Y1 - 2016/8/16
N2 - Incidence and mortality rates of anal cancer are increasing globally. More than 90% of anal squamous cell carcinomas (ASCC) are associated with human papillomavirus (HPV). Studies on HPV-related anogenital lesions have shown that patterns of methylation of viral and cellular DNA targets could potentially be developed as disease biomarkers. Lesion-specific DNA isolated from formalin-fixed paraffin-embedded (FFPE) tissues from existing or prospective patient cohorts may constitute a valuable resource for methylation analysis. However, low concentrations of DNA make these samples technically challenging to analyse using existing methods. We therefore set out to develop a sensitive and reproducible nested PCR-pyrosequencing based method to accurately quantify methylation at 10 CpG sites within the E2BS1, E2BS2,3,4 and Sp1 binding sites in the viral upstream regulatory region of HPV16 genome. Methylation analyses using primary and nested PCR-pyrosequencing on 52 FFPE tissue [26 paired whole tissue sections (WTS) and laser capture microdissected (LCM) tissues] from patients with anal squamous intraepithelial lesions was performed. Using nested PCR, methylation results were obtained for the E2BS1, E2BS2,3,4 and Sp1 binding sites in 86.4% of the WTS and 81.8% of the LCM samples. Methylation patterns were strongly correlated within median values of matched pairs of WTS and LCM sections, but overall methylation was higher in LCM samples at different CpG sites. High grade lesions showed low methylation levels in the E2BS1 and E2BS2 regions, with increased methylation detected in the E2BS,3,4/Sp1 regions, showing the highest methylation at CpG site 37. The method developed is highly sensitive in samples with low amounts of DNA and demonstrated to be suitable for archival samples. Our data shows a possible role of specific methylation in the HPV16 URR for detection of HSIL.
AB - Incidence and mortality rates of anal cancer are increasing globally. More than 90% of anal squamous cell carcinomas (ASCC) are associated with human papillomavirus (HPV). Studies on HPV-related anogenital lesions have shown that patterns of methylation of viral and cellular DNA targets could potentially be developed as disease biomarkers. Lesion-specific DNA isolated from formalin-fixed paraffin-embedded (FFPE) tissues from existing or prospective patient cohorts may constitute a valuable resource for methylation analysis. However, low concentrations of DNA make these samples technically challenging to analyse using existing methods. We therefore set out to develop a sensitive and reproducible nested PCR-pyrosequencing based method to accurately quantify methylation at 10 CpG sites within the E2BS1, E2BS2,3,4 and Sp1 binding sites in the viral upstream regulatory region of HPV16 genome. Methylation analyses using primary and nested PCR-pyrosequencing on 52 FFPE tissue [26 paired whole tissue sections (WTS) and laser capture microdissected (LCM) tissues] from patients with anal squamous intraepithelial lesions was performed. Using nested PCR, methylation results were obtained for the E2BS1, E2BS2,3,4 and Sp1 binding sites in 86.4% of the WTS and 81.8% of the LCM samples. Methylation patterns were strongly correlated within median values of matched pairs of WTS and LCM sections, but overall methylation was higher in LCM samples at different CpG sites. High grade lesions showed low methylation levels in the E2BS1 and E2BS2 regions, with increased methylation detected in the E2BS,3,4/Sp1 regions, showing the highest methylation at CpG site 37. The method developed is highly sensitive in samples with low amounts of DNA and demonstrated to be suitable for archival samples. Our data shows a possible role of specific methylation in the HPV16 URR for detection of HSIL.
UR - http://www.scopus.com/inward/record.url?scp=84984817811&partnerID=8YFLogxK
UR - http://purl.org/au-research/grants/nhmrc/568971
UR - http://purl.org/au-research/grants/nhmrc/1071269
U2 - 10.1371/journal.pone.0160673
DO - 10.1371/journal.pone.0160673
M3 - Article
C2 - 27529629
AN - SCOPUS:84984817811
SN - 1932-6203
VL - 11
SP - 1
EP - 15
JO - PLoS ONE
JF - PLoS ONE
IS - 8
M1 - e0160673
ER -