- Email: [email protected]
Increased methylation of Human Papillomavirus type 16 DNA correlates with viral integration in Vulval Intraepithelial Neoplasia
Journal of Clinical Virology 61 (2014) 393–399
Contents lists available at ScienceDirect
Journal of Clinical Virology journal homepage: www.elsevier.com/locate/jcv
Increased methylation of Human Papillomavirus type 16 DNA correlates with viral integration in Vulval Intraepithelial Neoplasia Dean Bryant, Tiffany Onions, Rachel Raybould, Sadie Jones, Amanda Tristram, Samantha Hibbitts, Alison Fiander, Ned Powell ∗ HPV Research Group, Institute of Cancer and Genetics, School of Medicine, Cardiff University, Heath Park, Cardiff CF14 4XN, UK
a r t i c l e
i n f o
Article history: Received 20 June 2014 Received in revised form 25 July 2014 Accepted 8 August 2014 Keywords: Human papillomavirus HPV Vulval Intraepithelial Neoplasia Methylation Integration
a b s t r a c t Background: Methylation of HPV16 DNA is a promising biomarker for triage of HPV positive cervical screening samples but the biological basis for the association between HPV-associated neoplasia and increased methylation is unclear. Objectives: To determine whether HPV16 DNA methylation was associated with viral integration, and investigate the relationships between viral DNA methylation, integration and gene expression. Study design: HPV16 DNA methylation, integration and gene expression were assessed using pyrosequencing, ligation-mediated PCR and QPCR, in biopsies from 25 patients attending a specialist vulval neoplasia clinic and in short-term clonal cell lines derived from vulval and vaginal neoplasia. Results: Increased methylation of the HPV16 L1/L2 and E2 regions was associated with integration of viral DNA into the host genome. This relationship was observed both in vivo and in vitro. Increased methylation of E2 binding sites did not appear to be associated with greater expression of viral early genes. Expression of HPV E6 and E7 did not correlate with either integration state or increased L1/L2 methylation. Conclusions: The data suggest that increased HPV DNA methylation may be partly attributable to viral integration, and provide a biological rationale for quantification of L1/L2 methylation in triage of HPV positive cervical screening samples. © 2014 Elsevier B.V. All rights reserved.
1. Background Human papillomavirus (HPV) is the underlying cause of >99% of cervical cancers, and substantial proportions of anal, vulval, vaginal and oropharyngeal cancers [1–3]. HPV type 16 alone is responsible for 61% of cervical cancers and 67% of Vulval Intraepithelial Neoplasia (VIN) [4,5]. HPV testing is likely to replace cytology as the primary screen for cervical neoplasia [6], however the majority of HPV infections are transient and clinically inconsequential, hence a method to identify clinically significant infections is urgently required. Methylation of HPV16 DNA correlates with disease grade [7–11], and has shown considerable promise as a triage marker in preliminary clinical studies [10,12,13]. Previous studies have
Abbreviations: HPV, human papillomavirus; LCR, Long Control Region; DIPS, Detection of Integrated Papillomavirus Sequences; APOT, Amplification of Papillomavirus Oncogene Transcripts; E2BS, E2 binding site. ∗ Corresponding author. Tel.: +44 02920 744742; fax: +44 2920 744399. E-mail addresses: [email protected] (D. Bryant), [email protected] (T. Onions), [email protected] (R. Raybould), [email protected] (S. Jones), [email protected] (A. Tristram), [email protected] (S. Hibbitts), fi[email protected] (A. Fiander), [email protected] (N. Powell). http://dx.doi.org/10.1016/j.jcv.2014.08.006 1386-6532/© 2014 Elsevier B.V. All rights reserved.
established the importance of integration of viral DNA into the host genome [14] and of up-regulation of the E6 and E7 oncogenes in driving neoplasia [15], but how viral DNA methylation relates to these aspects of viral oncogenesis remains to be established. The HPV16 p97 early promoter, located at the 3 end of the Long Control Region (LCR), governs transcription of the early genes responsible for viral replication and cellular proliferation. During productive HPV infections, transcription is tightly controlled by the binding of cellular transcription factors and the viral E2 gene product within the LCR in a differentiation dependent manner [16]. Integration of HPV DNA into the host genome frequently results in loss of E2 expression, relieving repression of the p97 promoter and leading to cellular transformation [14], although greater expression of HPV16 E6 and E7 has not been associated with HPV16 integration in vivo [17], and 28% of cervical cancers do not contain integrated HPV [18]. Some studies have reported E2 binding site (E2BS) methylation in association with cervical neoplasia and cancer, and suggest that methylation of E2BS might prevent E2 binding, resulting in increased expression of the E6 and E7 ORFs [7,19,20]. We hypothesised that in instances without integration-mediated loss of E2 expression, changes in DNA methylation might constitute an alternative mechanism of transformation.
394
D. Bryant et al. / Journal of Clinical Virology 61 (2014) 393–399
2. Objectives The current investigation addressed four main hypotheses: (1) HPV integration is associated with hypermethylation of HPV L1/L2 regions; (2) increased methylation of E2BS will be present in samples without integrated HPV; (3) hypermethylation of the L1/L2 region is associated with higher levels of E6/E7 expression; and (4) HPV integration is associated with higher levels of E6/E7 expression. To test these hypotheses, HPV16 methylation, integration and gene expression were investigated in a cohort of 25 predominantly VIN patients. VIN is a precursor lesion of invasive vulval carcinoma, and a painful, distressing and chronic condition which causes considerable psychosexual morbidity [21,22]. The VIN cohort was selected as a high proportion of VIN patients are positive for HPV16 [23] and integration occurs in around 40% of high grade VIN [24]. To complement the in vivo analyses, methylation, integration and gene expression were also determined in short-term cell cultures derived from VIN and Vaginal Intraepithelial Neoplasia (VaIN) biopsies. 3. Study design 3.1. Clinical samples and cell lines Following ethics committee approval (SMKW/EL/03/5178 and 08/RPM/4250), biopsies were obtained with informed consent from patients attending a specialist VIN clinic, and stored in ThinPrep (Bedford, USA) prior to nucleic acid extraction. Twenty-five HPV16 positive biopsies were tested, including twenty-two cases of VIN3, one VaIN3, one Anal Intraepithelial Neoplasia (AIN2) and one vulval biopsy without discernible VIN. Histopathology was performed by a consultant pathologist with special interest in VIN. Patient age ranged from 22 to 81 years (mean 45.3). Biopsies were transported in Thinprep liquid based cytology media (Hologic, Bedford, USA), then homogenised using a TissueRuptor (Qiagen, Hilden, Germany), and nucleic acids extracted using RNeasy (Fibrous Tissue) and QIAamp DNA extraction kits (Qiagen). Short-term cell lines were established from primary cultures and grown as described by Stanley et al. [25]. CU-VI-8 was derived from a 46-year-old woman with VIN3. CU-VA-9 was derived from a 31-year-old woman with VAIN3. Three distinct clonal lines were derived from each biopsy via single cell cloning (described in Bryant et al. [26]). Nucleic acids were extracted using the AllPrep DNA/RNA kit (Qiagen) according the manufacturer’s instructions. 3.2. HPV DNA typing and integration analyses HPV infection was identified using the PapilloCheck® assay (Greiner Bio-One GmbH, Frickenhausen, Germany), as described previously [23]. Integrated HPV DNA was identified using Detection of Integrated Papillomavirus Sequences (DIPS) [27], with primer sequences described by Thorland et al. [28]. HPV–human mRNA fusion transcripts were detected using Amplification of Papillomavirus Oncogene Transcripts (APOT) [29]. Following DIPS and APOT, PCR products were electrophoresed, excised and sequenced. PCR using primers flanking integration sites confirmed the presence of the integration event in the original DNA and cDNA. Due to the small amounts of RNA recovered from clinical biopsies, APOT was only performed on cell line material. Amplification of fulllength HPV16 E2 DNA was performed as described previously [30]. 3.3. HPV DNA methylation and gene expression analyses DNA (500 ng) was sodium bisulfite treated using the EZ-DNA methylation kit (Zymo Research Corp, CA, USA). DNA methylation was assessed by pyrosequencing the E2 ORF, L1/L2 overlap,
Table 1 Summary of integration events detected by DIPS in biopsy material. Patient #
Histology
Age
HPV junction regiona
Integration locib
24 32 37 39 42 44 46 53
VIN3 VIN3 VIN3 VIN3 VIN3 VIN3 VaIN3 VIN3
45 30 60 39 51 54 38 57
1540 (E1) 3348 (E4) 3161 (E2) 1786 (E1) 4910 (L2) 3166 (E2) 2508 (E1) 4989 (L2)
8q24.21 2q35 1q32.1 1p36.22 Satellite DNAc 17q24.3 9q21.31 3q26.31
a Indicates the location of the HPV:Human junction in HPV DNA (nucleotide numbering relates to NC001526.1). b Indicates the location of the HPV:Human junction in human DNA. c Homology was found to HSATII satellite DNA in Chr.7, Chr.22, Chr.2, Chr.16, Chr.10 and Chr.Y.
enhancer and promoter regions (Qiagen PyroMark Q96 ID) as described by Bryant et al. [11]. The assays each target multiple CpGs and were performed in duplicate. This assay has been shown to be highly reproducible when assessed in multiple independent runs with HPV16 positive CaSki cell line DNA (Supplementary Figure 1). Methylation levels are reported as means for each region.mRNA levels were assessed by Q-RT-PCR with assays specific for E2, E4, E6 and E7 (N.B. the E4 QPCR amplifies both E2 and E4 transcripts). Two reference genes were used (TBP and HPRT) and relative quantification was performed using Qbase Plus Software (Biogazelle, Ghent) [31]. Primers and reaction conditions for pyrosequencing and QPCR are listed in Supplementary Table 1. 3.4. Statistical methods Distributions of values between groups were compared using independent samples Mann–Whitney U tests. Correlations were assessed using Spearman’s coefficient. Analyses were performed in IBM SPSS Statistics 20. 4. Results HPV DNA integration, methylation and gene expression were assessed by DIPS, pyrosequencing and QPCR in 25 HPV16 positive biopsies. Fig. 1 shows HPV16 genome organisation and location of assays. Integrated HPV was detected in 8/25 (32%) samples (Table 1). Integration disrupted several HPV ORFs including E1, E2, E4 and L2. The median ages for patient with and without HPV integration were 48.0 vs. 44.0 years (p = 0.475). DNA methylation varied considerably both among the regions of the HPV genome and among patients (Fig. 2). Methylation of the promoter and enhancer regions was consistently low in all samples (range 1.0–4.7% and 0.0–4.3% respectively) whilst E2 and L1/L2 methylation ranged from 1.2–89.7% and 1.6–88.0% respectively. DNA methylation in the E2 and/or L1/L2 regions appeared to be higher in samples in which HPV integration was identified. In the L1/L2 region, methylation levels were significantly higher in samples with evidence of integrated HPV (p = 0.011). This relationship was not universal though, as two samples (VR32 and VR42) displayed integration but showed E2 and/or L1/L2 methylation levels similar to samples without integrated HPV. We hypothesised that where integration was not observed, increased methylation of inhibitory E2BS might occur. However methylation levels in the main inhibitory E2BS did not differ according to HPV integration (p = 0.344). A similar result was obtained for the enhancer region (p = 0.475). E6/E7 mRNA levels varied considerably among samples (Fig. 2), but were not significantly correlated with L1/L2, or E2BS (promoter) methylation. Similarly there was no significant difference in E6/E7 mRNA levels between samples with high (>50%) L1/L2
D. Bryant et al. / Journal of Clinical Virology 61 (2014) 393–399
395
Fig. 1. HPV16 genome and location of assays. Upper panel: HPV16 genome organisation showing ORFs (grey arrows), pyrosequencing assays (filled black boxes) and QPCR amplimers (unfilled grey boxes). Lower panel: Schematic representation of CpG and E2BS in the HPV16 LCR. The high affinity (activation) BS is shown in black, the noncanonical site is striped and the low affinity (repressive) sites are in grey. White boxes show the location of pyrosequencing amplimers. Numbering shows nucleotide position relative to NC001526.1.
methylation versus low methylation (Fisher’s test: E6 p = 0.869, E7 p = 0.303), and there were no significant differences in E6/E7 gene expression between samples with and without integration (p = 0.711, p = 0.315). Unsurprisingly, E2/E4 mRNA transcript levels were significantly lower in samples in which integration was identified (p = 0.04). Investigation of gene expression, DNA methylation and integration in patient samples is essential to ensure that conclusions are relevant to clinical disease; however some observations can be confounded by the heterogeneous nature of biopsy material, e.g. assessment of HPV gene expression can be problematic when samples contain a proportion of uninfected epithelium. These variables were therefore also assessed in short-term VIN- and VaIN-derived clonal cell lines. HPV integration in the clonal lines was assessed by DIPS, by PCR for full-length E2 DNA, and by APOT. Distinct integration events were detected in clones CU-VA-9D, CU-VA-9H, CU-VI-8M and CUVI-8Y (Table 2). Integration events were not detected in CU-VA-9A or CU-VI-8P, however CU-VI-8P carried a deletion of the E2 ORF and given the well-characterised roles of E2 protein in viral replication, genome maintenance, partition and tethering [32], it appears likely that CU-VI-8P contained integrated HPV (the presence of integrated HPV in this line was also consistent with mRNA sequencing data [26]). In most lines, DIPS and APOT identified integration in the same chromosomal location, but in CU-VA-9H the two techniques detected integration in distinct locations. These results were consistent between early and late passages and suggest integration of HPV at multiple sites. In CU-VI-8Y, the presence of amplifiable full-length E2 DNA in conjunction with disruption of this region detected by DIPS, suggests the presence of mixed episomal and integrated HPV or of concatenated insertions of full-length HPV. DNA methylation varied considerably both among individual clones and also with passage (Fig. 3). As observed with patient samples, clones with integrated HPV showed greater methylation especially in the E2 and/or L1/L2 regions. Clone CU-VA-9A, in which integrated HPV was not detected, showed lowest L1/L2 methylation. Stable, high levels of methylation in the L1/L2 region (defined
as >50% in all passages) were present in two of the six clones (CU-VI8M and Y), and both these lines showed below average expression of HPV E6 and E7 relative to the other four clones. However dynamic changes in L1/L2 methylation that correlated with gene expression were evident in CU-VA-9D, where increasing methylation in the L1/L2 region correlated significantly with increasing E4 and E6 (p = <0.01). This line expressed relatively high levels of all HPV transcripts examined, and showed the lowest levels of E2 and promoter methylation. An inverse relationship between LCR methylation and transcription was also observed in CU-VI-8Y, which showed the highest levels of LCR methylation (>20%) and lowest E6/E7 transcription. Gene expression did not appear to be related to integration status. Variation in E6/E7 transcript levels was observed among the lines independent of integration state, and the lines without confirmed integration (CU-VA-9A and CU-VI-8P) showed approximately average levels of E6 and E7 transcripts.
5. Discussion The main finding of this study was the apparent association between integration of HPV DNA into the host genome and methylation of the L1/L2 and E2 regions. This relationship was observed both in vivo and in vitro. We did not observe convincing evidence to support the hypotheses that changes to methylation patterns in the LCR constitute an alternative to HPV integration, or resulted in increased E6/E7 gene expression. Neither hypermethylation of the L1/L2 region, nor HPV integration, appeared to be associated with increased transcription of HPV E6/E7. Methylation of HPV16 L1 and L2 DNA correlates with severity of cervical neoplasia, and has considerable potential as a biomarker to augment HPV testing in primary cervical screening [11]. However the precise biological basis for the relationship between L1/L2 methylation and disease progression is unclear. Integration of HPV into the host genome is present in the majority of cervical cancers [18], and may be significant for several reasons: loss of expression
396
D. Bryant et al. / Journal of Clinical Virology 61 (2014) 393–399
Fig. 2. HPV16 DNA methylation and gene expression in VIN biopsies. Methylation in the four regions assayed is shown in the panels on the left. Relative gene expression is shown in the panels on the right. Note differences in scales between upper and lower panels. Error bars denote standard error.
of the E2 protein may result in upregulation of the E6 and E7 oncogenes [33]; integration may be associated with genomic instability and amplification of oncogenes [18]; or it may simply prevent loss of viral episomes due to antiviral responses. Unfortunately, direct assessment of HPV integration is technically challenging and is unlikely to be widely applicable to clinical samples. The current data suggest that increased methylation of L1/L2 is associated with HPV integration. The specific methylation of
HPV DNA may arise via de novo methylation-mediated silencing of foreign DNA [34]; by activation of an instructive programme of methylation imposed by HPV gene expression [35]; or as a self-regulatory mechanism to prevent excessive oncogene expression [36]. Methylation of the viral late regions has also been suggested to be an indicator of the duration of an infection in a given tissue, due to the up-regulation of DNA methyltransferases (DNMT) caused by HPV gene expression [35], and so may
Table 2 HPV integration status in short-term clonal cell lines derived from CU-VA-9 and CU-VI-8. Biopsy
CU-VA-9
CU-VI-8
Cell line (passages)
A (5,11) D (5,11) H (5,11) M (5,9) P (5,9) Y (9,13)
E2 PCRa
Intact Intact Intact Disrupted Disrupted Intact
DIPSd
APOTd
HPV junctionb
Integration locic
HPV junctionb
Integration locic
ND 5003 (L2) 2490 (E1) 1194 (E1) 2201 (E1)–3147 (E2) 3167 (E2)
ND 18p11.31 11p15.3 3q28 ND 3p21.31
ND 3716 (E4) 882 (E1) 880 (E1) ND 882 (E1)
ND 18p11.31 5q11.2 3q28 ND 3p21.31
ND: not detected. a Indicates whether full length E2 could be amplified from DNA. b Indicates the location of the HPV:Human fusion in HPV (nucleotide numbering relates to NC001526.1). c Indicates the chromosomal location at which integration was identified. d All integration sites listed were present in DNA/RNA from early and late passage cultures.
D. Bryant et al. / Journal of Clinical Virology 61 (2014) 393–399
397
Fig. 3. HPV16 DNA methylation and gene expression in VIN and VAIN clonal cell lines. Left panels shows mean methylation at the four regions assayed with increasing passage (5–6 passages) for CU-VI-8- and CU-VA-9-derived clonal lines. Right panel shows relative gene expression with increasing passage for CU-VI-8- and CU-VA-9-derived clonal lines. If no data are shown, this indicates transcripts were not detected. Error bars denote standard error. Presence of integrated HPV was confirmed in lines 9D, 9H, 8M and 8Y.
also act as a proxy for viral persistence and disease progression [37]. Our data contrast with a study of methylation, gene expression and integration based on the W12 cell line [38], in which increased L1 methylation was not associated with HPV integration. However a later study from the same group observed an association between integration and increased methylation in cervical cytology samples from 21 patients attending a colposcopy clinic (albeit measured non-quantitatively by bisulphite sequencing for four L1 CpGs) [39]. One of our initial hypotheses was that hypermethylation of E2BS could block binding of the E2 protein and act as an alternative transforming event to integration [40,41]. If this were correct, neoplasia associated with episomal infections might show increased methylation of E2BS relative to integrated infections i.e. the transforming methylation pattern and the presence of integration would be mutually exclusive. In this study, there was no significant difference in promoter methylation between episomal and integrated infections in vivo or in vitro; similarly, no such differences were observed when individual CpGs were considered rather than mean values (data not shown). Other studies have reported E2BS methylation in association with cervical cancers, although more rarely in high grade precancerous lesions [7,19,20,42], and it has
been suggested that LCR E2BSs need not be heavily methylated in order to influence P97 activity [43,44]. This apparent contrast may be method dependent because pyrosequencing considers the mean methylation of a mix of DNAs, if each DNA were variably methylated in one or two E2BS CpGs the overall level of methylation might be low, but most genomes might be repressed. Alternatively, E2BS hypermethylation could be an early and transient event. As L1/L2 DNA methylation has previously been shown to be associated with higher grade disease [7,11,45], we investigated whether L1/L2 methylation level was associated with E6/E7 oncogene expression. We found no evidence for higher levels of E6/E7 expression in association with increased L1/L2 methylation. As might be expected, there appeared to be correlation between transcription of full-length E2 and HPV integration in patient samples, but consistent with previous studies, there was no discernable correlation between integration and E6 or E7 expression [17]. This strengths of the study included use of both a clinical cohort and short-term clonal cell lines derived from VIN and VAIN; observations that are consistent between the two systems are more likely to be generally applicable. Furthermore, this consistency suggests that the in vitro models provide a reasonable representation of in vivo mechanisms. Use of pyrosequencing was a significant
398
D. Bryant et al. / Journal of Clinical Virology 61 (2014) 393–399
strength as it provides a quantitative assessment of methylation for multiple CpGs in a heterogeneous mix of DNAs. Potential limitations included use of DIPS to detect integration, as it is not quantitative and does not show whether a particular integrant is transcriptionally active. Furthermore, both DIPS and APOT may have limited sensitivity in samples where detection of integration would require amplification of very large (several Kb) fragments, which might explain why integration was not identified in CU-VI8P. However, taken together, the DIPS and transcription data appear likely to give a broadly accurate indication of HPV integration state. We acknowledge that the relatively modest sample size limits the statistical power of the study and recognise that our conclusions require confirmation in a larger cohort. We conclude that assessment of HPV DNA methylation is a promising pragmatic biomarker to aid screening for HPVassociated cervical neoplasia, especially in the context of triage of HPV positive samples [46]. It is important to understand the biological basis for increased methylation in order to understand the likely limitations of this methodology. Our data provide a biological rationale for quantification of L1/L2 methylation in triage of HPV positive cervical samples and imply that increased methylation may be partly attributable to viral integration, which has previously been suggested to be a key event in the carcinogenetic process [14]. Finally, the CU-VI-8 and CU-VA-9 clonal cell lines may provide a useful model for more thorough investigation of the relationships between HPV DNA methylation, integration and gene expression. Funding This work was supported by a Cancer Research Wales Fellowship awarded to DB and by a Welsh Government NISCHR Studentship awarded to TO. Competing interests None declared. Ethical approval Research Ethics Committee approval was obtained (Refs SMKW/EL/03/5178 and 08/RPM/4250). Acknowledgments We are very grateful to Cancer Research Wales and NISCHR (HS09016) for funding; to Triantafillos Liloglou (Liverpool University) for help with methylation assay design; and to Mandy Gilkes for assistance with pyrosequencing. Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jcv.2014.08.006. References [1] De Vuyst H, Clifford GM, Nascimento MC, Madeleine MM, Franceschi S. Prevalence and type distribution of human papillomavirus in carcinoma and intraepithelial neoplasia of the vulva, vagina and anus: a meta-analysis. Int J Cancer 2009;124:1626–36. [2] Mehanna H, Beech T, Nicholson T, El-Hariry I, McConkey C, Paleri V, et al. Prevalence of human papillomavirus in oropharyngeal and nonoropharyngeal head and neck cancer – systematic review and meta-analysis of trends by time and region. Head & Neck 2012;35:747–55. [3] Walboomers JM, Jacobs MV, Manos MM, Bosch FX, Kummer JA, Shah KV, et al. Human papillomavirus is a necessary cause of invasive cervical cancer worldwide. J Pathol 1999;189:12–9.
[4] de Sanjose S, Alemany L, Ordi J, Tous S, Alejo M, Bigby SM, et al. Worldwide human papillomavirus genotype attribution in over 2000 cases of intraepithelial and invasive lesions of the vulva. Eur J Cancer 2013;49:3450–61. [5] de Sanjose S, Quint WG, Alemany L, Geraets DT, Klaustermeier JE, Lloveras B, et al. Human papillomavirus genotype attribution in invasive cervical cancer: a retrospective cross-sectional worldwide study. Lancet Oncol 2010;11:1048–56. [6] Hesselink AT, Heideman DA, Steenbergen RD, Coupe VM, Overmeer RM, Rijkaart D, et al. Combined promoter methylation analysis of CADM1 and MAL: an objective triage tool for high-risk human papillomavirus DNA-positive women. Clin Cancer Res 2011;17:2459–65. [7] Fernandez AF, Rosales C, Lopez-Nieva P, Grana O, Ballestar E, Ropero S, et al. The dynamic DNA methylomes of double-stranded DNA viruses associated with human cancer. Genome Res 2009;19:438–51. [8] Kalantari M, Garcia-Carranca A, Morales-Vazquez CD, Zuna R, Montiel DP, Calleja-Macias IE, et al. Laser capture microdissection of cervical human papillomavirus infections: copy number of the virus in cancerous and normal tissue and heterogeneous DNA methylation. Virology 2009;390:261–7. [9] Sun C, Reimers LL, Burk RD. Methylation of HPV16 genome CpG sites is associated with cervix precancer and cancer. Gynecol Oncol 2011;121:59–63. [10] Mirabello L, Sun C, Ghosh A, Rodriguez AC, Schiffman M, Wentzensen N, et al. Methylation of human papillomavirus type 16 genome and risk of cervical precancer in a Costa Rican population. J Nat Cancer Inst 2012;104:556–65. [11] Bryant D, Tristram A, Liloglou T, Hibbitts S, Fiander A, Powell N. Quantitative measurement of Human Papillomavirus type 16 L1/L2 DNA methylation correlates with cervical disease grade. J Clin Virol 2014;59:24–9. [12] Lorincz AT, Brentnall AR, Vasiljevic N, Scibior-Bentkowska D, Castanon A, Fiander A, et al. HPV16 L1 and L2 DNA methylation predicts high-grade cervical intraepithelial neoplasia in women with mildly abnormal cervical cytology. Int J Cancer 2013;133:637–44. [13] Mirabello L, Schiffman M, Ghosh A, Rodriguez AC, Vasiljevic N, Wentzensen N, et al. Elevated methylation of HPV16 DNA is associated with the development of high grade cervical intraepithelial neoplasia. Int J Cancer 2013;132: 1412–22. [14] Pett M, Coleman N. Integration of high-risk human papillomavirus: a key event in cervical carcinogenesis? J Pathol 2007;212:356–67. [15] Moody CA, Laimins LA. Human papillomavirus oncoproteins: pathways to transformation. Nat Rev Cancer 2010;10:550–60. [16] Thierry F. Transcriptional regulation of the papillomavirus oncogenes by cellular and viral transcription factors in cervical carcinoma. Virology 2009;384:375–9. [17] Hafner N, Driesch C, Gajda M, Jansen L, Kirchmayr R, Runnebaum IB, et al. Integration of the HPV16 genome does not invariably result in high levels of viral oncogene transcripts. Oncogene 2008;27:1610–7. [18] Ojesina AI, Lichtenstein L, Freeman SS, Pedamallu CS, Imaz-Rosshandler I, Pugh TJ, et al. Landscape of genomic alterations in cervical carcinomas. Nature 2014;506:371–5. [19] Bhattacharjee B, Sengupta S. CpG methylation of HPV 16 LCR at E2 binding site proximal to P97 is associated with cervical cancer in presence of intact E2. Virology 2006;354:280–5. [20] Snellenberg S, Schütze DM, Claassen-Kramer D, Meijer CJLM, Snijders PJF, Steenbergen RDM. Methylation status of the E2 binding sites of HPV16 in cervical lesions determined with the Luminex® xMAPTM system. Virology 2012;422:357–65. [21] Likes WM, Stegbauer C, Tillmanns T, Pruett J. Correlates of sexual function following vulvar excision. Gynecol Oncol 2007;105:600–3. [22] Shylasree TS, Karanjgaokar V, Tristram A, Wilkes AR, MacLean AB, Fiander AN. Contribution of demographic, psychological and disease-related factors to quality of life in women with high-grade vulval intraepithelial neoplasia. Gynecol Oncol 2008;110:185–9. [23] Bryant D, Rai N, Rowlands G, Hibbitts S, Jones J, Tristram A, et al. Human papillomavirus type distribution in vulval intraepithelial neoplasia determined using PapilloCheck DNA Microarray. J Med Virol 2011;83:1358–61. [24] Hillemanns P, Wang X. Integration of HPV-16 and HPV-18 DNA in vulvar intraepithelial neoplasia. Gynecol Oncol 2006;100:276–82. [25] Stanley MA, Browne HM, Appleby M, Minson AC. Properties of a nontumorigenic human cervical keratinocyte cell line. Int J Cancer 1989;43:672–6. [26] Bryant D, Onions T, Raybould R, Flynn Á, Tristram A, Meyrick S, et al. mRNA sequencing of novel cell lines from Human Papillomavirus type-16 related vulval intraepithelial neoplasia: consequences of expression of HPV16 E4 and E5. J Med Virol 2014 June 5, http://dx.doi.org/10.1002/jmv.23994 [Epub ahead of print]. [27] Luft F, Klaes R, Nees M, Durst M, Heilmann V, Melsheimer P, et al. Detection of integrated papillomavirus sequences by ligation-mediated PCR (DIPS-PCR) and molecular characterization in cervical cancer cells. Int J Cancer 2001;92:9–17. [28] Thorland EC, Myers SL, Persing DH, Sarkar G, McGovern RM, Gostout BS, et al. Human papillomavirus type 16 integrations in cervical tumors frequently occur in common fragile sites. Cancer Res 2000;60:5916–21. [29] Klaes R, Woerner SM, Ridder R, Wentzensen N, Duerst M, Schneider A, et al. Detection of high-risk cervical intraepithelial neoplasia and cervical cancer by amplification of transcripts derived from integrated papillomavirus oncogenes. Cancer Res 1999;59:6132–6. [30] Collins SI, Constandinou-Williams C, Wen K, Young LS, Roberts S, Murray PG, et al. Disruption of the E2 gene is a common and early event in the natural history of cervical human papillomavirus infection: a longitudinal cohort study. Cancer Res 2009;69:3828–32.
D. Bryant et al. / Journal of Clinical Virology 61 (2014) 393–399 [31] Hellemans J, Mortier G, De Paepe A, Speleman F, Vandesompele J. qBase relative quantification framework and software for management and automated analysis of real-time quantitative PCR data. Genome Biol 2007;8:R19. [32] McBride AA. The papillomavirus E2 proteins. Virology 2013;445:57–79. [33] Jeon S, Allen-Hoffmann BL, Lambert PF. Integration of human papillomavirus type 16 into the human genome correlates with a selective growth advantage of cells. J Virol 1995;69:2989–97. [34] Heller H, Kammer C, Wilgenbus P, Doerfler W. Chromosomal insertion of foreign (adenovirus type 12, plasmid, or bacteriophage lambda) DNA is associated with enhanced methylation of cellular DNA segments. Proc Natl Acad Sci U S A 1995;92:5515–9. [35] Leonard SM, Wei W, Collins SI, Pereira M, Diyaf A, Constandinou-Williams C, et al. Oncogenic human papillomavirus imposes an instructive pattern of DNA methylation changes which parallel the natural history of cervical HPV infection in young women. Carcinogenesis 2012;33:1286–93. [36] De-Castro Arce J, Gockel-Krzikalla E, Rosl F. Silencing of multi-copy HPV16 by viral self-methylation and chromatin occlusion: a model for epigenetic virus–host interaction. Hum Mol Genet 2012;21:1693–705. [37] Lorincz AT. Cancer diagnostic classifiers based on quantitative DNA methylation. Expert Rev Mol Diagn 2014;14:293–305. [38] Kalantari M, Lee D, Calleja-Macias IE, Lambert PF, Bernard HU. Effects of cellular differentiation, chromosomal integration and 5-aza-2 -deoxycytidine treatment on human papillomavirus-16 DNA methylation in cultured cell lines. Virology 2008;374:292–303. [39] Kalantari M, Chase DM, Tewari KS, Bernard HU. Recombination of human papillomavirus-16 and host DNA in exfoliated cervical cells: a pilot study of L1
[40]
[41]
[42]
[43]
[44]
[45]
[46]
399
gene methylation and chromosomal integration as biomarkers of carcinogenic progression. J Med Virol 2010;82:311–20. Thain A, Jenkins O, Clarke AR, Gaston K. CpG methylation directly inhibits binding of the human papillomavirus type 16 E2 protein to specific DNA sequences. J Virol 1996;70:7233–5. Kim K, Garner-Hamrick PA, Fisher C, Lee D, Lambert PF. Methylation patterns of papillomavirus DNA, its influence on E2 function, and implications in viral infection. J Virol 2003;77:12450–9. Jacquin E, Baraquin A, Ramanah R, Carcopino X, Morel A, Valmary-Degano S, et al. Methylation of human papillomavirus type 16 CpG sites at E2binding site 1 (E2BS1), E2BS2, and the Sp1-binding site in cervical cancer samples as determined by high-resolution melting analysis-PCR. J Clin Microbiol 2013;51:3207–15. Stunkel W, Bernard HU. The chromatin structure of the long control region of human papillomavirus type 16 represses viral oncoprotein expression. J Virol 1999;73:1918–30. Kalantari M, Villa LL, Calleja-Macias IE, Bernard HU. Human papillomavirus-16 and -18 in penile carcinomas: DNA methylation, chromosomal recombination and genomic variation. Int J Cancer 2008;123:1832–40. Brandsma JL, Sun Y, Lizardi PM, Tuck DP, Zelterman D, Haines GK3rd, et al. Distinct human papillomavirus type 16 methylomes in cervical cells at different stages of premalignancy. Virology 2009;389:100–7. Clarke MA, Wentzensen N, Mirabello L, Ghosh A, Wacholder S, Harari A, et al. Human papillomavirus DNA methylation as a potential biomarker for cervical cancer. Cancer Epidemiol Biomarkers Prev 2012;21:2125–37.
Contents lists available at ScienceDirect
Journal of Clinical Virology journal homepage: www.elsevier.com/locate/jcv
Increased methylation of Human Papillomavirus type 16 DNA correlates with viral integration in Vulval Intraepithelial Neoplasia Dean Bryant, Tiffany Onions, Rachel Raybould, Sadie Jones, Amanda Tristram, Samantha Hibbitts, Alison Fiander, Ned Powell ∗ HPV Research Group, Institute of Cancer and Genetics, School of Medicine, Cardiff University, Heath Park, Cardiff CF14 4XN, UK
a r t i c l e
i n f o
Article history: Received 20 June 2014 Received in revised form 25 July 2014 Accepted 8 August 2014 Keywords: Human papillomavirus HPV Vulval Intraepithelial Neoplasia Methylation Integration
a b s t r a c t Background: Methylation of HPV16 DNA is a promising biomarker for triage of HPV positive cervical screening samples but the biological basis for the association between HPV-associated neoplasia and increased methylation is unclear. Objectives: To determine whether HPV16 DNA methylation was associated with viral integration, and investigate the relationships between viral DNA methylation, integration and gene expression. Study design: HPV16 DNA methylation, integration and gene expression were assessed using pyrosequencing, ligation-mediated PCR and QPCR, in biopsies from 25 patients attending a specialist vulval neoplasia clinic and in short-term clonal cell lines derived from vulval and vaginal neoplasia. Results: Increased methylation of the HPV16 L1/L2 and E2 regions was associated with integration of viral DNA into the host genome. This relationship was observed both in vivo and in vitro. Increased methylation of E2 binding sites did not appear to be associated with greater expression of viral early genes. Expression of HPV E6 and E7 did not correlate with either integration state or increased L1/L2 methylation. Conclusions: The data suggest that increased HPV DNA methylation may be partly attributable to viral integration, and provide a biological rationale for quantification of L1/L2 methylation in triage of HPV positive cervical screening samples. © 2014 Elsevier B.V. All rights reserved.
1. Background Human papillomavirus (HPV) is the underlying cause of >99% of cervical cancers, and substantial proportions of anal, vulval, vaginal and oropharyngeal cancers [1–3]. HPV type 16 alone is responsible for 61% of cervical cancers and 67% of Vulval Intraepithelial Neoplasia (VIN) [4,5]. HPV testing is likely to replace cytology as the primary screen for cervical neoplasia [6], however the majority of HPV infections are transient and clinically inconsequential, hence a method to identify clinically significant infections is urgently required. Methylation of HPV16 DNA correlates with disease grade [7–11], and has shown considerable promise as a triage marker in preliminary clinical studies [10,12,13]. Previous studies have
Abbreviations: HPV, human papillomavirus; LCR, Long Control Region; DIPS, Detection of Integrated Papillomavirus Sequences; APOT, Amplification of Papillomavirus Oncogene Transcripts; E2BS, E2 binding site. ∗ Corresponding author. Tel.: +44 02920 744742; fax: +44 2920 744399. E-mail addresses: [email protected] (D. Bryant), [email protected] (T. Onions), [email protected] (R. Raybould), [email protected] (S. Jones), [email protected] (A. Tristram), [email protected] (S. Hibbitts), fi[email protected] (A. Fiander), [email protected] (N. Powell). http://dx.doi.org/10.1016/j.jcv.2014.08.006 1386-6532/© 2014 Elsevier B.V. All rights reserved.
established the importance of integration of viral DNA into the host genome [14] and of up-regulation of the E6 and E7 oncogenes in driving neoplasia [15], but how viral DNA methylation relates to these aspects of viral oncogenesis remains to be established. The HPV16 p97 early promoter, located at the 3 end of the Long Control Region (LCR), governs transcription of the early genes responsible for viral replication and cellular proliferation. During productive HPV infections, transcription is tightly controlled by the binding of cellular transcription factors and the viral E2 gene product within the LCR in a differentiation dependent manner [16]. Integration of HPV DNA into the host genome frequently results in loss of E2 expression, relieving repression of the p97 promoter and leading to cellular transformation [14], although greater expression of HPV16 E6 and E7 has not been associated with HPV16 integration in vivo [17], and 28% of cervical cancers do not contain integrated HPV [18]. Some studies have reported E2 binding site (E2BS) methylation in association with cervical neoplasia and cancer, and suggest that methylation of E2BS might prevent E2 binding, resulting in increased expression of the E6 and E7 ORFs [7,19,20]. We hypothesised that in instances without integration-mediated loss of E2 expression, changes in DNA methylation might constitute an alternative mechanism of transformation.
394
D. Bryant et al. / Journal of Clinical Virology 61 (2014) 393–399
2. Objectives The current investigation addressed four main hypotheses: (1) HPV integration is associated with hypermethylation of HPV L1/L2 regions; (2) increased methylation of E2BS will be present in samples without integrated HPV; (3) hypermethylation of the L1/L2 region is associated with higher levels of E6/E7 expression; and (4) HPV integration is associated with higher levels of E6/E7 expression. To test these hypotheses, HPV16 methylation, integration and gene expression were investigated in a cohort of 25 predominantly VIN patients. VIN is a precursor lesion of invasive vulval carcinoma, and a painful, distressing and chronic condition which causes considerable psychosexual morbidity [21,22]. The VIN cohort was selected as a high proportion of VIN patients are positive for HPV16 [23] and integration occurs in around 40% of high grade VIN [24]. To complement the in vivo analyses, methylation, integration and gene expression were also determined in short-term cell cultures derived from VIN and Vaginal Intraepithelial Neoplasia (VaIN) biopsies. 3. Study design 3.1. Clinical samples and cell lines Following ethics committee approval (SMKW/EL/03/5178 and 08/RPM/4250), biopsies were obtained with informed consent from patients attending a specialist VIN clinic, and stored in ThinPrep (Bedford, USA) prior to nucleic acid extraction. Twenty-five HPV16 positive biopsies were tested, including twenty-two cases of VIN3, one VaIN3, one Anal Intraepithelial Neoplasia (AIN2) and one vulval biopsy without discernible VIN. Histopathology was performed by a consultant pathologist with special interest in VIN. Patient age ranged from 22 to 81 years (mean 45.3). Biopsies were transported in Thinprep liquid based cytology media (Hologic, Bedford, USA), then homogenised using a TissueRuptor (Qiagen, Hilden, Germany), and nucleic acids extracted using RNeasy (Fibrous Tissue) and QIAamp DNA extraction kits (Qiagen). Short-term cell lines were established from primary cultures and grown as described by Stanley et al. [25]. CU-VI-8 was derived from a 46-year-old woman with VIN3. CU-VA-9 was derived from a 31-year-old woman with VAIN3. Three distinct clonal lines were derived from each biopsy via single cell cloning (described in Bryant et al. [26]). Nucleic acids were extracted using the AllPrep DNA/RNA kit (Qiagen) according the manufacturer’s instructions. 3.2. HPV DNA typing and integration analyses HPV infection was identified using the PapilloCheck® assay (Greiner Bio-One GmbH, Frickenhausen, Germany), as described previously [23]. Integrated HPV DNA was identified using Detection of Integrated Papillomavirus Sequences (DIPS) [27], with primer sequences described by Thorland et al. [28]. HPV–human mRNA fusion transcripts were detected using Amplification of Papillomavirus Oncogene Transcripts (APOT) [29]. Following DIPS and APOT, PCR products were electrophoresed, excised and sequenced. PCR using primers flanking integration sites confirmed the presence of the integration event in the original DNA and cDNA. Due to the small amounts of RNA recovered from clinical biopsies, APOT was only performed on cell line material. Amplification of fulllength HPV16 E2 DNA was performed as described previously [30]. 3.3. HPV DNA methylation and gene expression analyses DNA (500 ng) was sodium bisulfite treated using the EZ-DNA methylation kit (Zymo Research Corp, CA, USA). DNA methylation was assessed by pyrosequencing the E2 ORF, L1/L2 overlap,
Table 1 Summary of integration events detected by DIPS in biopsy material. Patient #
Histology
Age
HPV junction regiona
Integration locib
24 32 37 39 42 44 46 53
VIN3 VIN3 VIN3 VIN3 VIN3 VIN3 VaIN3 VIN3
45 30 60 39 51 54 38 57
1540 (E1) 3348 (E4) 3161 (E2) 1786 (E1) 4910 (L2) 3166 (E2) 2508 (E1) 4989 (L2)
8q24.21 2q35 1q32.1 1p36.22 Satellite DNAc 17q24.3 9q21.31 3q26.31
a Indicates the location of the HPV:Human junction in HPV DNA (nucleotide numbering relates to NC001526.1). b Indicates the location of the HPV:Human junction in human DNA. c Homology was found to HSATII satellite DNA in Chr.7, Chr.22, Chr.2, Chr.16, Chr.10 and Chr.Y.
enhancer and promoter regions (Qiagen PyroMark Q96 ID) as described by Bryant et al. [11]. The assays each target multiple CpGs and were performed in duplicate. This assay has been shown to be highly reproducible when assessed in multiple independent runs with HPV16 positive CaSki cell line DNA (Supplementary Figure 1). Methylation levels are reported as means for each region.mRNA levels were assessed by Q-RT-PCR with assays specific for E2, E4, E6 and E7 (N.B. the E4 QPCR amplifies both E2 and E4 transcripts). Two reference genes were used (TBP and HPRT) and relative quantification was performed using Qbase Plus Software (Biogazelle, Ghent) [31]. Primers and reaction conditions for pyrosequencing and QPCR are listed in Supplementary Table 1. 3.4. Statistical methods Distributions of values between groups were compared using independent samples Mann–Whitney U tests. Correlations were assessed using Spearman’s coefficient. Analyses were performed in IBM SPSS Statistics 20. 4. Results HPV DNA integration, methylation and gene expression were assessed by DIPS, pyrosequencing and QPCR in 25 HPV16 positive biopsies. Fig. 1 shows HPV16 genome organisation and location of assays. Integrated HPV was detected in 8/25 (32%) samples (Table 1). Integration disrupted several HPV ORFs including E1, E2, E4 and L2. The median ages for patient with and without HPV integration were 48.0 vs. 44.0 years (p = 0.475). DNA methylation varied considerably both among the regions of the HPV genome and among patients (Fig. 2). Methylation of the promoter and enhancer regions was consistently low in all samples (range 1.0–4.7% and 0.0–4.3% respectively) whilst E2 and L1/L2 methylation ranged from 1.2–89.7% and 1.6–88.0% respectively. DNA methylation in the E2 and/or L1/L2 regions appeared to be higher in samples in which HPV integration was identified. In the L1/L2 region, methylation levels were significantly higher in samples with evidence of integrated HPV (p = 0.011). This relationship was not universal though, as two samples (VR32 and VR42) displayed integration but showed E2 and/or L1/L2 methylation levels similar to samples without integrated HPV. We hypothesised that where integration was not observed, increased methylation of inhibitory E2BS might occur. However methylation levels in the main inhibitory E2BS did not differ according to HPV integration (p = 0.344). A similar result was obtained for the enhancer region (p = 0.475). E6/E7 mRNA levels varied considerably among samples (Fig. 2), but were not significantly correlated with L1/L2, or E2BS (promoter) methylation. Similarly there was no significant difference in E6/E7 mRNA levels between samples with high (>50%) L1/L2
D. Bryant et al. / Journal of Clinical Virology 61 (2014) 393–399
395
Fig. 1. HPV16 genome and location of assays. Upper panel: HPV16 genome organisation showing ORFs (grey arrows), pyrosequencing assays (filled black boxes) and QPCR amplimers (unfilled grey boxes). Lower panel: Schematic representation of CpG and E2BS in the HPV16 LCR. The high affinity (activation) BS is shown in black, the noncanonical site is striped and the low affinity (repressive) sites are in grey. White boxes show the location of pyrosequencing amplimers. Numbering shows nucleotide position relative to NC001526.1.
methylation versus low methylation (Fisher’s test: E6 p = 0.869, E7 p = 0.303), and there were no significant differences in E6/E7 gene expression between samples with and without integration (p = 0.711, p = 0.315). Unsurprisingly, E2/E4 mRNA transcript levels were significantly lower in samples in which integration was identified (p = 0.04). Investigation of gene expression, DNA methylation and integration in patient samples is essential to ensure that conclusions are relevant to clinical disease; however some observations can be confounded by the heterogeneous nature of biopsy material, e.g. assessment of HPV gene expression can be problematic when samples contain a proportion of uninfected epithelium. These variables were therefore also assessed in short-term VIN- and VaIN-derived clonal cell lines. HPV integration in the clonal lines was assessed by DIPS, by PCR for full-length E2 DNA, and by APOT. Distinct integration events were detected in clones CU-VA-9D, CU-VA-9H, CU-VI-8M and CUVI-8Y (Table 2). Integration events were not detected in CU-VA-9A or CU-VI-8P, however CU-VI-8P carried a deletion of the E2 ORF and given the well-characterised roles of E2 protein in viral replication, genome maintenance, partition and tethering [32], it appears likely that CU-VI-8P contained integrated HPV (the presence of integrated HPV in this line was also consistent with mRNA sequencing data [26]). In most lines, DIPS and APOT identified integration in the same chromosomal location, but in CU-VA-9H the two techniques detected integration in distinct locations. These results were consistent between early and late passages and suggest integration of HPV at multiple sites. In CU-VI-8Y, the presence of amplifiable full-length E2 DNA in conjunction with disruption of this region detected by DIPS, suggests the presence of mixed episomal and integrated HPV or of concatenated insertions of full-length HPV. DNA methylation varied considerably both among individual clones and also with passage (Fig. 3). As observed with patient samples, clones with integrated HPV showed greater methylation especially in the E2 and/or L1/L2 regions. Clone CU-VA-9A, in which integrated HPV was not detected, showed lowest L1/L2 methylation. Stable, high levels of methylation in the L1/L2 region (defined
as >50% in all passages) were present in two of the six clones (CU-VI8M and Y), and both these lines showed below average expression of HPV E6 and E7 relative to the other four clones. However dynamic changes in L1/L2 methylation that correlated with gene expression were evident in CU-VA-9D, where increasing methylation in the L1/L2 region correlated significantly with increasing E4 and E6 (p = <0.01). This line expressed relatively high levels of all HPV transcripts examined, and showed the lowest levels of E2 and promoter methylation. An inverse relationship between LCR methylation and transcription was also observed in CU-VI-8Y, which showed the highest levels of LCR methylation (>20%) and lowest E6/E7 transcription. Gene expression did not appear to be related to integration status. Variation in E6/E7 transcript levels was observed among the lines independent of integration state, and the lines without confirmed integration (CU-VA-9A and CU-VI-8P) showed approximately average levels of E6 and E7 transcripts.
5. Discussion The main finding of this study was the apparent association between integration of HPV DNA into the host genome and methylation of the L1/L2 and E2 regions. This relationship was observed both in vivo and in vitro. We did not observe convincing evidence to support the hypotheses that changes to methylation patterns in the LCR constitute an alternative to HPV integration, or resulted in increased E6/E7 gene expression. Neither hypermethylation of the L1/L2 region, nor HPV integration, appeared to be associated with increased transcription of HPV E6/E7. Methylation of HPV16 L1 and L2 DNA correlates with severity of cervical neoplasia, and has considerable potential as a biomarker to augment HPV testing in primary cervical screening [11]. However the precise biological basis for the relationship between L1/L2 methylation and disease progression is unclear. Integration of HPV into the host genome is present in the majority of cervical cancers [18], and may be significant for several reasons: loss of expression
396
D. Bryant et al. / Journal of Clinical Virology 61 (2014) 393–399
Fig. 2. HPV16 DNA methylation and gene expression in VIN biopsies. Methylation in the four regions assayed is shown in the panels on the left. Relative gene expression is shown in the panels on the right. Note differences in scales between upper and lower panels. Error bars denote standard error.
of the E2 protein may result in upregulation of the E6 and E7 oncogenes [33]; integration may be associated with genomic instability and amplification of oncogenes [18]; or it may simply prevent loss of viral episomes due to antiviral responses. Unfortunately, direct assessment of HPV integration is technically challenging and is unlikely to be widely applicable to clinical samples. The current data suggest that increased methylation of L1/L2 is associated with HPV integration. The specific methylation of
HPV DNA may arise via de novo methylation-mediated silencing of foreign DNA [34]; by activation of an instructive programme of methylation imposed by HPV gene expression [35]; or as a self-regulatory mechanism to prevent excessive oncogene expression [36]. Methylation of the viral late regions has also been suggested to be an indicator of the duration of an infection in a given tissue, due to the up-regulation of DNA methyltransferases (DNMT) caused by HPV gene expression [35], and so may
Table 2 HPV integration status in short-term clonal cell lines derived from CU-VA-9 and CU-VI-8. Biopsy
CU-VA-9
CU-VI-8
Cell line (passages)
A (5,11) D (5,11) H (5,11) M (5,9) P (5,9) Y (9,13)
E2 PCRa
Intact Intact Intact Disrupted Disrupted Intact
DIPSd
APOTd
HPV junctionb
Integration locic
HPV junctionb
Integration locic
ND 5003 (L2) 2490 (E1) 1194 (E1) 2201 (E1)–3147 (E2) 3167 (E2)
ND 18p11.31 11p15.3 3q28 ND 3p21.31
ND 3716 (E4) 882 (E1) 880 (E1) ND 882 (E1)
ND 18p11.31 5q11.2 3q28 ND 3p21.31
ND: not detected. a Indicates whether full length E2 could be amplified from DNA. b Indicates the location of the HPV:Human fusion in HPV (nucleotide numbering relates to NC001526.1). c Indicates the chromosomal location at which integration was identified. d All integration sites listed were present in DNA/RNA from early and late passage cultures.
D. Bryant et al. / Journal of Clinical Virology 61 (2014) 393–399
397
Fig. 3. HPV16 DNA methylation and gene expression in VIN and VAIN clonal cell lines. Left panels shows mean methylation at the four regions assayed with increasing passage (5–6 passages) for CU-VI-8- and CU-VA-9-derived clonal lines. Right panel shows relative gene expression with increasing passage for CU-VI-8- and CU-VA-9-derived clonal lines. If no data are shown, this indicates transcripts were not detected. Error bars denote standard error. Presence of integrated HPV was confirmed in lines 9D, 9H, 8M and 8Y.
also act as a proxy for viral persistence and disease progression [37]. Our data contrast with a study of methylation, gene expression and integration based on the W12 cell line [38], in which increased L1 methylation was not associated with HPV integration. However a later study from the same group observed an association between integration and increased methylation in cervical cytology samples from 21 patients attending a colposcopy clinic (albeit measured non-quantitatively by bisulphite sequencing for four L1 CpGs) [39]. One of our initial hypotheses was that hypermethylation of E2BS could block binding of the E2 protein and act as an alternative transforming event to integration [40,41]. If this were correct, neoplasia associated with episomal infections might show increased methylation of E2BS relative to integrated infections i.e. the transforming methylation pattern and the presence of integration would be mutually exclusive. In this study, there was no significant difference in promoter methylation between episomal and integrated infections in vivo or in vitro; similarly, no such differences were observed when individual CpGs were considered rather than mean values (data not shown). Other studies have reported E2BS methylation in association with cervical cancers, although more rarely in high grade precancerous lesions [7,19,20,42], and it has
been suggested that LCR E2BSs need not be heavily methylated in order to influence P97 activity [43,44]. This apparent contrast may be method dependent because pyrosequencing considers the mean methylation of a mix of DNAs, if each DNA were variably methylated in one or two E2BS CpGs the overall level of methylation might be low, but most genomes might be repressed. Alternatively, E2BS hypermethylation could be an early and transient event. As L1/L2 DNA methylation has previously been shown to be associated with higher grade disease [7,11,45], we investigated whether L1/L2 methylation level was associated with E6/E7 oncogene expression. We found no evidence for higher levels of E6/E7 expression in association with increased L1/L2 methylation. As might be expected, there appeared to be correlation between transcription of full-length E2 and HPV integration in patient samples, but consistent with previous studies, there was no discernable correlation between integration and E6 or E7 expression [17]. This strengths of the study included use of both a clinical cohort and short-term clonal cell lines derived from VIN and VAIN; observations that are consistent between the two systems are more likely to be generally applicable. Furthermore, this consistency suggests that the in vitro models provide a reasonable representation of in vivo mechanisms. Use of pyrosequencing was a significant
398
D. Bryant et al. / Journal of Clinical Virology 61 (2014) 393–399
strength as it provides a quantitative assessment of methylation for multiple CpGs in a heterogeneous mix of DNAs. Potential limitations included use of DIPS to detect integration, as it is not quantitative and does not show whether a particular integrant is transcriptionally active. Furthermore, both DIPS and APOT may have limited sensitivity in samples where detection of integration would require amplification of very large (several Kb) fragments, which might explain why integration was not identified in CU-VI8P. However, taken together, the DIPS and transcription data appear likely to give a broadly accurate indication of HPV integration state. We acknowledge that the relatively modest sample size limits the statistical power of the study and recognise that our conclusions require confirmation in a larger cohort. We conclude that assessment of HPV DNA methylation is a promising pragmatic biomarker to aid screening for HPVassociated cervical neoplasia, especially in the context of triage of HPV positive samples [46]. It is important to understand the biological basis for increased methylation in order to understand the likely limitations of this methodology. Our data provide a biological rationale for quantification of L1/L2 methylation in triage of HPV positive cervical samples and imply that increased methylation may be partly attributable to viral integration, which has previously been suggested to be a key event in the carcinogenetic process [14]. Finally, the CU-VI-8 and CU-VA-9 clonal cell lines may provide a useful model for more thorough investigation of the relationships between HPV DNA methylation, integration and gene expression. Funding This work was supported by a Cancer Research Wales Fellowship awarded to DB and by a Welsh Government NISCHR Studentship awarded to TO. Competing interests None declared. Ethical approval Research Ethics Committee approval was obtained (Refs SMKW/EL/03/5178 and 08/RPM/4250). Acknowledgments We are very grateful to Cancer Research Wales and NISCHR (HS09016) for funding; to Triantafillos Liloglou (Liverpool University) for help with methylation assay design; and to Mandy Gilkes for assistance with pyrosequencing. Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jcv.2014.08.006. References [1] De Vuyst H, Clifford GM, Nascimento MC, Madeleine MM, Franceschi S. Prevalence and type distribution of human papillomavirus in carcinoma and intraepithelial neoplasia of the vulva, vagina and anus: a meta-analysis. Int J Cancer 2009;124:1626–36. [2] Mehanna H, Beech T, Nicholson T, El-Hariry I, McConkey C, Paleri V, et al. Prevalence of human papillomavirus in oropharyngeal and nonoropharyngeal head and neck cancer – systematic review and meta-analysis of trends by time and region. Head & Neck 2012;35:747–55. [3] Walboomers JM, Jacobs MV, Manos MM, Bosch FX, Kummer JA, Shah KV, et al. Human papillomavirus is a necessary cause of invasive cervical cancer worldwide. J Pathol 1999;189:12–9.
[4] de Sanjose S, Alemany L, Ordi J, Tous S, Alejo M, Bigby SM, et al. Worldwide human papillomavirus genotype attribution in over 2000 cases of intraepithelial and invasive lesions of the vulva. Eur J Cancer 2013;49:3450–61. [5] de Sanjose S, Quint WG, Alemany L, Geraets DT, Klaustermeier JE, Lloveras B, et al. Human papillomavirus genotype attribution in invasive cervical cancer: a retrospective cross-sectional worldwide study. Lancet Oncol 2010;11:1048–56. [6] Hesselink AT, Heideman DA, Steenbergen RD, Coupe VM, Overmeer RM, Rijkaart D, et al. Combined promoter methylation analysis of CADM1 and MAL: an objective triage tool for high-risk human papillomavirus DNA-positive women. Clin Cancer Res 2011;17:2459–65. [7] Fernandez AF, Rosales C, Lopez-Nieva P, Grana O, Ballestar E, Ropero S, et al. The dynamic DNA methylomes of double-stranded DNA viruses associated with human cancer. Genome Res 2009;19:438–51. [8] Kalantari M, Garcia-Carranca A, Morales-Vazquez CD, Zuna R, Montiel DP, Calleja-Macias IE, et al. Laser capture microdissection of cervical human papillomavirus infections: copy number of the virus in cancerous and normal tissue and heterogeneous DNA methylation. Virology 2009;390:261–7. [9] Sun C, Reimers LL, Burk RD. Methylation of HPV16 genome CpG sites is associated with cervix precancer and cancer. Gynecol Oncol 2011;121:59–63. [10] Mirabello L, Sun C, Ghosh A, Rodriguez AC, Schiffman M, Wentzensen N, et al. Methylation of human papillomavirus type 16 genome and risk of cervical precancer in a Costa Rican population. J Nat Cancer Inst 2012;104:556–65. [11] Bryant D, Tristram A, Liloglou T, Hibbitts S, Fiander A, Powell N. Quantitative measurement of Human Papillomavirus type 16 L1/L2 DNA methylation correlates with cervical disease grade. J Clin Virol 2014;59:24–9. [12] Lorincz AT, Brentnall AR, Vasiljevic N, Scibior-Bentkowska D, Castanon A, Fiander A, et al. HPV16 L1 and L2 DNA methylation predicts high-grade cervical intraepithelial neoplasia in women with mildly abnormal cervical cytology. Int J Cancer 2013;133:637–44. [13] Mirabello L, Schiffman M, Ghosh A, Rodriguez AC, Vasiljevic N, Wentzensen N, et al. Elevated methylation of HPV16 DNA is associated with the development of high grade cervical intraepithelial neoplasia. Int J Cancer 2013;132: 1412–22. [14] Pett M, Coleman N. Integration of high-risk human papillomavirus: a key event in cervical carcinogenesis? J Pathol 2007;212:356–67. [15] Moody CA, Laimins LA. Human papillomavirus oncoproteins: pathways to transformation. Nat Rev Cancer 2010;10:550–60. [16] Thierry F. Transcriptional regulation of the papillomavirus oncogenes by cellular and viral transcription factors in cervical carcinoma. Virology 2009;384:375–9. [17] Hafner N, Driesch C, Gajda M, Jansen L, Kirchmayr R, Runnebaum IB, et al. Integration of the HPV16 genome does not invariably result in high levels of viral oncogene transcripts. Oncogene 2008;27:1610–7. [18] Ojesina AI, Lichtenstein L, Freeman SS, Pedamallu CS, Imaz-Rosshandler I, Pugh TJ, et al. Landscape of genomic alterations in cervical carcinomas. Nature 2014;506:371–5. [19] Bhattacharjee B, Sengupta S. CpG methylation of HPV 16 LCR at E2 binding site proximal to P97 is associated with cervical cancer in presence of intact E2. Virology 2006;354:280–5. [20] Snellenberg S, Schütze DM, Claassen-Kramer D, Meijer CJLM, Snijders PJF, Steenbergen RDM. Methylation status of the E2 binding sites of HPV16 in cervical lesions determined with the Luminex® xMAPTM system. Virology 2012;422:357–65. [21] Likes WM, Stegbauer C, Tillmanns T, Pruett J. Correlates of sexual function following vulvar excision. Gynecol Oncol 2007;105:600–3. [22] Shylasree TS, Karanjgaokar V, Tristram A, Wilkes AR, MacLean AB, Fiander AN. Contribution of demographic, psychological and disease-related factors to quality of life in women with high-grade vulval intraepithelial neoplasia. Gynecol Oncol 2008;110:185–9. [23] Bryant D, Rai N, Rowlands G, Hibbitts S, Jones J, Tristram A, et al. Human papillomavirus type distribution in vulval intraepithelial neoplasia determined using PapilloCheck DNA Microarray. J Med Virol 2011;83:1358–61. [24] Hillemanns P, Wang X. Integration of HPV-16 and HPV-18 DNA in vulvar intraepithelial neoplasia. Gynecol Oncol 2006;100:276–82. [25] Stanley MA, Browne HM, Appleby M, Minson AC. Properties of a nontumorigenic human cervical keratinocyte cell line. Int J Cancer 1989;43:672–6. [26] Bryant D, Onions T, Raybould R, Flynn Á, Tristram A, Meyrick S, et al. mRNA sequencing of novel cell lines from Human Papillomavirus type-16 related vulval intraepithelial neoplasia: consequences of expression of HPV16 E4 and E5. J Med Virol 2014 June 5, http://dx.doi.org/10.1002/jmv.23994 [Epub ahead of print]. [27] Luft F, Klaes R, Nees M, Durst M, Heilmann V, Melsheimer P, et al. Detection of integrated papillomavirus sequences by ligation-mediated PCR (DIPS-PCR) and molecular characterization in cervical cancer cells. Int J Cancer 2001;92:9–17. [28] Thorland EC, Myers SL, Persing DH, Sarkar G, McGovern RM, Gostout BS, et al. Human papillomavirus type 16 integrations in cervical tumors frequently occur in common fragile sites. Cancer Res 2000;60:5916–21. [29] Klaes R, Woerner SM, Ridder R, Wentzensen N, Duerst M, Schneider A, et al. Detection of high-risk cervical intraepithelial neoplasia and cervical cancer by amplification of transcripts derived from integrated papillomavirus oncogenes. Cancer Res 1999;59:6132–6. [30] Collins SI, Constandinou-Williams C, Wen K, Young LS, Roberts S, Murray PG, et al. Disruption of the E2 gene is a common and early event in the natural history of cervical human papillomavirus infection: a longitudinal cohort study. Cancer Res 2009;69:3828–32.
D. Bryant et al. / Journal of Clinical Virology 61 (2014) 393–399 [31] Hellemans J, Mortier G, De Paepe A, Speleman F, Vandesompele J. qBase relative quantification framework and software for management and automated analysis of real-time quantitative PCR data. Genome Biol 2007;8:R19. [32] McBride AA. The papillomavirus E2 proteins. Virology 2013;445:57–79. [33] Jeon S, Allen-Hoffmann BL, Lambert PF. Integration of human papillomavirus type 16 into the human genome correlates with a selective growth advantage of cells. J Virol 1995;69:2989–97. [34] Heller H, Kammer C, Wilgenbus P, Doerfler W. Chromosomal insertion of foreign (adenovirus type 12, plasmid, or bacteriophage lambda) DNA is associated with enhanced methylation of cellular DNA segments. Proc Natl Acad Sci U S A 1995;92:5515–9. [35] Leonard SM, Wei W, Collins SI, Pereira M, Diyaf A, Constandinou-Williams C, et al. Oncogenic human papillomavirus imposes an instructive pattern of DNA methylation changes which parallel the natural history of cervical HPV infection in young women. Carcinogenesis 2012;33:1286–93. [36] De-Castro Arce J, Gockel-Krzikalla E, Rosl F. Silencing of multi-copy HPV16 by viral self-methylation and chromatin occlusion: a model for epigenetic virus–host interaction. Hum Mol Genet 2012;21:1693–705. [37] Lorincz AT. Cancer diagnostic classifiers based on quantitative DNA methylation. Expert Rev Mol Diagn 2014;14:293–305. [38] Kalantari M, Lee D, Calleja-Macias IE, Lambert PF, Bernard HU. Effects of cellular differentiation, chromosomal integration and 5-aza-2 -deoxycytidine treatment on human papillomavirus-16 DNA methylation in cultured cell lines. Virology 2008;374:292–303. [39] Kalantari M, Chase DM, Tewari KS, Bernard HU. Recombination of human papillomavirus-16 and host DNA in exfoliated cervical cells: a pilot study of L1
[40]
[41]
[42]
[43]
[44]
[45]
[46]
399
gene methylation and chromosomal integration as biomarkers of carcinogenic progression. J Med Virol 2010;82:311–20. Thain A, Jenkins O, Clarke AR, Gaston K. CpG methylation directly inhibits binding of the human papillomavirus type 16 E2 protein to specific DNA sequences. J Virol 1996;70:7233–5. Kim K, Garner-Hamrick PA, Fisher C, Lee D, Lambert PF. Methylation patterns of papillomavirus DNA, its influence on E2 function, and implications in viral infection. J Virol 2003;77:12450–9. Jacquin E, Baraquin A, Ramanah R, Carcopino X, Morel A, Valmary-Degano S, et al. Methylation of human papillomavirus type 16 CpG sites at E2binding site 1 (E2BS1), E2BS2, and the Sp1-binding site in cervical cancer samples as determined by high-resolution melting analysis-PCR. J Clin Microbiol 2013;51:3207–15. Stunkel W, Bernard HU. The chromatin structure of the long control region of human papillomavirus type 16 represses viral oncoprotein expression. J Virol 1999;73:1918–30. Kalantari M, Villa LL, Calleja-Macias IE, Bernard HU. Human papillomavirus-16 and -18 in penile carcinomas: DNA methylation, chromosomal recombination and genomic variation. Int J Cancer 2008;123:1832–40. Brandsma JL, Sun Y, Lizardi PM, Tuck DP, Zelterman D, Haines GK3rd, et al. Distinct human papillomavirus type 16 methylomes in cervical cells at different stages of premalignancy. Virology 2009;389:100–7. Clarke MA, Wentzensen N, Mirabello L, Ghosh A, Wacholder S, Harari A, et al. Human papillomavirus DNA methylation as a potential biomarker for cervical cancer. Cancer Epidemiol Biomarkers Prev 2012;21:2125–37.