CpG methylation analysis of hpv16 in laser capture microdissected archival tissue and whole tissue sections from high grade anal squamous intraepithelial lesions: a potential disease biomarker

Monica Molano, Sepehr N. Tabrizi, Suzanne M. Garland, Jennifer M. Roberts, Dorothy A. MacHalek, Samuel Phillips, David Chandler, Richard J. Hillman, Andrew E. Grulich, Fengyi Jin, I. Mary Poynten, David J. Templeton, Alyssa M. Cornall, SPANC Study Team, Kirsten McCaffery, Kirsten Howard, Annabelle Farnsworth, Kit Fairley, Garrett Prestage, Andrew Carr & 19 others Winnie Tong, Carmella Law, Brian Acraman, Leonie Crampton, Lance Feeney, Eddie Fraissard, Matthew Law, Patrick McGrath, Robert Mellor, Richard Norris, Matthew O'Dwyer, Susan Pendlebury, Kathy Petoumenos, Adele Richards, Lance Schema, Daniel Seeds, Eva Segelov, Julia Thurloe, Rick Varma

Research output: Contribution to journalArticleResearchpeer-review

Abstract

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.

LanguageEnglish
Article numbere0160673
Pages1-15
Number of pages15
JournalPLoS ONE
Volume11
Issue number8
DOIs
Publication statusPublished - 16 Aug 2016

Fingerprint

Methylation
Biomarkers
lesions (animal)
methylation
lasers
biomarkers
Lasers
Tissue
Papillomaviridae
DNA
formalin
Paraffin
Polymerase Chain Reaction
alkanes
Formaldehyde
binding sites
sampling
tissues
Squamous Intraepithelial Lesions of the Cervix
Binding Sites

Bibliographical note

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.

Cite this

Molano, Monica ; Tabrizi, Sepehr N. ; Garland, Suzanne M. ; Roberts, Jennifer M. ; MacHalek, Dorothy A. ; Phillips, Samuel ; Chandler, David ; Hillman, Richard J. ; Grulich, Andrew E. ; Jin, Fengyi ; Poynten, I. Mary ; Templeton, David J. ; Cornall, Alyssa M. ; SPANC Study Team. / CpG methylation analysis of hpv16 in laser capture microdissected archival tissue and whole tissue sections from high grade anal squamous intraepithelial lesions : a potential disease biomarker. In: PLoS ONE. 2016 ; Vol. 11, No. 8. pp. 1-15.
@article{01947c57e3cc47aa95b0395cc6269529,
title = "CpG methylation analysis of hpv16 in laser capture microdissected archival tissue and whole tissue sections from high grade anal squamous intraepithelial lesions: a potential disease biomarker",
abstract = "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.",
author = "Monica Molano and Tabrizi, {Sepehr N.} and Garland, {Suzanne M.} and Roberts, {Jennifer M.} and MacHalek, {Dorothy A.} and Samuel Phillips and David Chandler and Hillman, {Richard J.} and Grulich, {Andrew E.} and Fengyi Jin and Poynten, {I. Mary} and Templeton, {David J.} and Cornall, {Alyssa M.} and {SPANC Study Team} and Kirsten McCaffery and Kirsten Howard and Annabelle Farnsworth and Kit Fairley and Garrett Prestage and Andrew Carr and Winnie Tong and Carmella Law and Brian Acraman and Leonie Crampton and Lance Feeney and Eddie Fraissard and Matthew Law and Patrick McGrath and Robert Mellor and Richard Norris and Matthew O'Dwyer and Susan Pendlebury and Kathy Petoumenos and Adele Richards and Lance Schema and Daniel Seeds and Eva Segelov and Julia Thurloe and Rick Varma",
note = "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.",
year = "2016",
month = "8",
day = "16",
doi = "10.1371/journal.pone.0160673",
language = "English",
volume = "11",
pages = "1--15",
journal = "PLoS ONE",
issn = "1932-6203",
publisher = "Public Library of Science",
number = "8",

}

Molano, M, Tabrizi, SN, Garland, SM, Roberts, JM, MacHalek, DA, Phillips, S, Chandler, D, Hillman, RJ, Grulich, AE, Jin, F, Poynten, IM, Templeton, DJ, Cornall, AM & SPANC Study Team 2016, 'CpG methylation analysis of hpv16 in laser capture microdissected archival tissue and whole tissue sections from high grade anal squamous intraepithelial lesions: a potential disease biomarker', PLoS ONE, vol. 11, no. 8, e0160673, pp. 1-15. https://doi.org/10.1371/journal.pone.0160673

CpG methylation analysis of hpv16 in laser capture microdissected archival tissue and whole tissue sections from high grade anal squamous intraepithelial lesions : a potential disease biomarker. / Molano, Monica; Tabrizi, Sepehr N.; Garland, Suzanne M.; Roberts, Jennifer M.; MacHalek, Dorothy A.; Phillips, Samuel; Chandler, David; Hillman, Richard J.; Grulich, Andrew E.; Jin, Fengyi; Poynten, I. Mary; Templeton, David J.; Cornall, Alyssa M.; SPANC Study Team.

In: PLoS ONE, Vol. 11, No. 8, e0160673, 16.08.2016, p. 1-15.

Research output: Contribution to journalArticleResearchpeer-review

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 - PLoS ONE

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

VL - 11

SP - 1

EP - 15

JO - PLoS ONE

JF - PLoS ONE

SN - 1932-6203

IS - 8

M1 - e0160673

ER -