Abstract
Despite the rising incidence, currently, there are no early detection methods for HPV-driven HNC (HPV-HNC). Cervical cancer studies suggest that HPV DNA methylation changes can be used as a biomarker to discriminate cancer patients from HPV-infected individuals. As such, this study was designed to establish a protocol to evaluate DNA methylation changes in HPV late genes and long control region (LCR) in saliva samples of HPV-HNC patients and HPV-positive controls. Higher methylation levels were detected in HPV late genes (L1 and L2) in both tumour and saliva samples of HPV-HNC patients compared with HPV-positive controls. Moreover, methylation patterns between tumours and corresponding saliva samples were observed to have a strong correlation (Passing-Bablok regression analysis; τ = 0.7483, P < 0.0001). Considering the differences between HNC and controls in methylation levels in late genes, and considering primer amplification efficiencies, 13 CpG sites located at L1 and L2 genes were selected for further evaluation. A total of 18 HNC saliva samples and 10 control saliva samples were assessed for the methylation levels in the selected sites. From the CpG sites evaluated statistically significant differences were identified for CpG sites at L2—CpG 6 (P = 0.0004), L1—CpG 3 (P = 0.0144), L1—CpG 2 (P = 0.0395) and L2—CpG 19 (P = 0.0455). Our pilot data indicate that higher levels of DNA methylation in HPV late genes are indicative of HPV-HNC risk, and it is a potential supplementary biomarker for salivary HPV detection-based HPV-HNC screening.
This study indicates that higher levels of DNA methylation in HPV late genes are indicative of HPV-HNC risk, and it is a potential supplementary biomarker for salivary HPV detection-based HPV-HNC screening.
Graphical Abstract
Graphical Abstract.
Introduction
Human papillomavirus (HPV) infection is a well-known risk factor for Head and Neck cancers (HNC). Despite the rising incidence of HPV-driven HNC (HPV-HNC), especially in the Western world, currently, there are no screening programs or early detection methods for HPV-HNC (1). Consequently, HPV-HNC is often diagnosed in advanced stages. Although HPV-HNC, particularly, those arising in the oropharynx are associated with a prognostic advantage, late diagnosis coerces patients to undergo extensive multi-modal treatment regimens often leading to treatment-associated morbidities (2).
Among the early detection methods proposed, salivary high-risk HPV detection-based longitudinal monitoring can be identified as the most promising method thus far (3–5). Due to the non-invasive nature and convenience of collection, saliva is an ideal specimen for screening purposes (6–9). Moreover, HPV testing is relatively inexpensive and required facilities are available in most settings. Despite these advantages, the proposed approach for HPV-HNC detection based only on salivary HPV testing is a prolonged process requiring long-term follow-ups (3,4). Since only a small proportion of salivary HPV-positive individuals develop HNC, a general consensus regarding the feasibility of salivary HPV testing as a screening technique for HPV-HNC has not been reached (10).
However, the feasibility of HPV-HNC detection can be improved if there are additional biomarkers to stratify the risk of developing cancer in HPV-positive individuals. One such biomarker proposed by cervical cancer studies is HPV DNA methylation. Unlike the human genome, HPV genome does not contain CpG clusters in the promoters or in gene bodies except for the dispersed CpG dinucleotides across the genome (11). However, studies suggest that methylation changes in the HPV early promoter region, particularly in the CpG dinucleotides positioned in HPV E2 binding sites, may promote oncogenesis (11–13). As E2 functions as a regulatory protein, inhibition of its promoter binding may upregulate oncoprotein expression increasing the odds of oncogenesis (11–13). Moreover, cervical cancer studies have reported that methylation changes in HPV late genes can discriminate patients with high-grade cervical lesions and cervical cancer from controls (14,15).
Although limited data is available, the HPV DNA methylation changes between HPV-HNC and their corresponding controls have not been investigated in detail (11,16). Due to the unavailability of a known pre-cancer, the methodologies applied by cervical cancer studies are not directly transferable to HPV-HNC studies (17). However, the ability to isolate HPV DNA from the saliva of these patients and controls provides an alternative avenue for assessing these associations. Hence this pilot study was designed to establish a protocol to evaluate DNA methylation changes in HPV late genes and long coding region (LCR) using saliva and to evaluate and compare HPV DNA methylation patterns between HPV-HNC patients and controls.
Materials and methods
Ethical considerations
Ethical clearance was obtained from Metro South Human Research Ethics Committee [HREC/12/QPAH/381]. Approval was also obtained from the University of Queensland Medical Ethical Institutional Board [HREC No: 2014000862], Royal Brisbane and Women’s Hospital (RBWH) [HREC/16/QRBW/447] and Queensland University of Technology [HREC No: 1400000617, 1400000641 and 200000043], University of Queensland Medical Ethical Institutional Board.
Participant recruitment
Samples were collected between 2012 and 2021 in Queensland, Australia. HNC patients were recruited from Royal Brisbane and Women’s Hospital, Princess Alexandra Hospital and Logan Hospital. HPV-positive controls were recruited from Logan Hospital, the Queensland University of Technology Health Clinics, the University of Queensland School of Dentistry and Metro-North Sexual Health and HIV Service.
Specimen collection
Surgically resected tumour tissues were obtained from the pathology department of the relevant hospital. Samples were transported on dry ice and processed for DNA isolation immediately. Saliva collection and processing were carried out according to our previously published methods (5,9). Samples were transported on ice and stored at −80°C.
DNA isolation
QIAamp® DNA Mini kit (QIAGEN, MD, USA) was used for DNA isolation. Fresh frozen tissue specimens were mechanically disrupted by grinding in liquid nitrogen and DNA isolation was carried out according to the manufacturer’s protocol. Similarly, DNA was isolated from whole saliva (200 µl) as per the manufacturer’s protocol (9). The recommended RNA digestion step was also followed to ensure the complete removal of genomic RNA.
HPV 16 testing
DNA isolated from saliva samples and tissue samples were tested for HPV16 by quantitative PCR. Detailed procedure is described elsewhere (6).
Bisulphite conversion
Isolated genomic DNA (0.5–2 µg) was subjected to bisulphite modification using EpiTect® Bisulphite conversion kit (QIAGEN, MD, USA) according to the manufacturer’s protocol. Briefly, bisulphite conversion reaction mixture was prepared by adding 85 µl of bisulphite solution, 35 µl DNA protect buffer and genomic DNA (up to 2 µg) and RNase-free water to adjust the total volume to 140 µl. The conversion was carried out in a C1000 Touch™ Thermal Cycler (Bio-Rad Inc., CA, USA) in three consecutive incubation steps for 25, 85 and 175 min at 60°C, each starting with an initial 5-min denaturation step. Following desulphonation and purification converted DNA was isolated using column filtration.
Quantitative DNA methylation assessment
EpiTYPER® assay (Agena Bioscience, CA, USA) was used for the quantitative detection of DNA methylation in HPV16 L1, L2 genes and LCR. The assay includes three steps where bisulphite converted DNA is amplified by PCR, dephosphorylating the remaining unincorporated dNTPs by Shrimp alkaline phosphatase (SAP) and MassCLEAVE reaction where reverse transcription and uracil-specific cleavage takes place. The final reaction mixture is analysed by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS).
Primer design
Bisulphite-conversion-based PCR Primers were designed using EpiDesigner platform (AgenaCx.com, Agena Bioscience, CA, USA). Eighteen pairs of primers were designed to cover either forward or reverse strands of HPV16 L1, L2 genes and LCR (Supplementary Table 1, available at Carcinogenesis Online). Reverse primers were designed to include a tag containing a T7 promoter sequence which is required at the MassCLEAVE step.
Amplification of bisulphite-converted DNA
Bisulphite-converted DNA was amplified by PCR—EmeraldAmp® GT (Takara Bio Inc., CA, USA) PCR master mix was used, and PCR amplification was carried out in a C1000 Touch™ Thermal Cycler. The total volume of PCR mixture was 25 µl including 12.5 µl of EmeraldAmp® GT master mixture, 0.25 µl each 100 nM forward and reverse primer and 60 ng of bisulphite converted DNA. The volume was adjusted using HPLC-grade water. Thermocycling conditions were 95°C for 4 min initial denaturation, 35 cycles each with 95°C for 30 s, optimal annealing temperature (Supplementary Table 1, available at Carcinogenesis Online) for 30 s, 72°C for 1 min and a final extension step at 72°C for 5 min.
De-phosphorylation
10 µl of the amplified sample was used for the de-phosphorylation reaction to inactivate the remaining additional dNTPs in the mixture. SAP cocktail was prepared by mixing 0.6 µl of SAP and 3.4 µl of HPLC-grade water and 4 µl of the SAP cocktail was used in each reaction. The reaction mixture was incubated at 37°C for 20 min and SAP was inactivated by incubating at 85°C for 5 min.
MassCLEAVE reaction
2 µl of the reaction mixture was used for in vitro RNA transcription and uracil-specific cleavage in mass cleave reaction. The composition of the MassCLEAVE cocktail is listed in Table 1 and 5 µl of the cocktail was added to each sample. The mixture was incubated for 3 h at 37°C.
Table 1.
The composition of MassCLEAVE cocktail
| Reaction component | Volume |
|---|---|
| HPLC-grade water | 3.21 µl |
| 5× T7 polymerase buffer | 0.89 µl |
| T cleavage mix | 0.22 µl |
| DTT, 100 mM | 0.22 µl |
| T7 RNA and DNA polymerase | 0.40 µl |
| RNase A | 0.06 µl |
| Total volume | 5.0 l |
MALDI-TOF MS
Following the addition of 41 µl of HPLC-grade water and conditioning with ion exchange resin, reaction mixtures were loaded into a SpectroCHIP Array and analysed using MassARRAY® Analyzer (Agena Bioscience, CA, USA).
Data analysis
MassARRAY® data was analysed by EpiTYPER software (Version 1.3) (Agena Bioscience CA, USA). Statistical analysis was performed using JMP Pro software version 17.0.0 (SAS Institute, Cary, NC, USA). The linearity and calibration between tumour and salivary HPV DNA methylation levels was analysed using Passing-Bablok regression and Bland-Altman paired analysis. Exact Mann–Whitney tests were used for the comparison of methylation patterns between HPV-HNC and controls.
Results
Participant information
Tumour samples (n = 8, fresh frozen), 18 saliva samples from HPV-HNC patients, (all together 24 HPV-HNC patients) and 10 saliva samples from HPV-positive controls were included in the study (Table 2). All the saliva samples from HNC patients were collected at the point of diagnosis prior to treatment.
Table 2.
Demographic and clinical characteristics of the study participants
| HPV-positive HNC (N = 21) | HPV-positive controls (N = 10) | ||
|---|---|---|---|
| Age | ≤55 | 2 (9.52%) | 4 (40.0%) |
| 56–65 | 16 (76.19%) | 3 (30.0%) | |
| ≥66 | 3 (14.29%) | 3 (30.0%) | |
| Gender | Male | 21 (100%) | 9 (90.0%) |
| Female | 0 (0%) | 1 (10.0%) | |
| HNC site | Tonsil | 15 (71.43%) | — |
| BOT | 3 (14.29%) | ||
| Tonsil and BOT | 2 (9.52%) | ||
| Floor of the mouth | 1 (4.76%) | ||
| AJCC stage (8th Edition) | Stage 01 | 8 (38.09%) | — |
| Stage 02 | 9 (42.85%) | ||
| Stage 03 | 2 (9.52%) | ||
| Stage 04 | 1 (4.76%) | ||
| Not available | 1 (4.76%) |
DNA methylation across HPV late genes and LCR
Six fresh-frozen HPV-HNC tumour samples were evaluated comprehensively for DNA methylation changes across HPV16 L1, L2 genes and LCR. Methylation levels ranged from complete methylation to no methylation in different CpG sites and methylation levels differ among samples. Despite the variations, higher levels of methylation were observed for several CpG sites in L1 and L2 genes compared with LCR region (Figure 1, Supplementary Table 2, available at Carcinogenesis Online).
Figure 1.
DNA methylation changes in CpG sites across HPV16 L1, L2 genes and LCR in HNC tissue (N = 6). Some of the primers designed for the reverse strand are overlapping and therefore, methylation values are indicated as reverser strand 1 and reverse strand 2.
Salivary detection of HPV DNA methylation
Two saliva samples each from HPV-HNC patients and HPV-positive controls were investigated for DNA methylation changes across HPV16 L1, L2 genes and LCR. Similar to HNC tissue samples, higher methylation levels were observed for several CpG sites in L1 and L2 genes in HNC saliva samples. However, methylation level of these sites was comparatively lower in HPV-positive control saliva samples (Figure 2, Supplementary Table 2, available at Carcinogenesis Online).
Figure 2.
DNA methylation changes in CpG sites across HPV16 L1, L2 genes and LCR in HNC saliva (N = 2) and HPV-positive controls’ saliva (N = 2). Some of the primers designed for the reverse strand are overlapping and therefore, methylation values are indicated as reverser strand 1 and reverse strand 2.
The concordance between HNC tissue and HNC salivary for HPV DNA methylation
We conducted an investigation to assess the concordance of HPV DNA methylation patterns between salivary HPV DNA and tumour HPV DNA, aiming to evaluate the extent to which saliva accurately represents tumour HPV DNA methylation. Five HNC tumour tissue samples and their corresponding saliva samples were considered and methylation data for 99 CpG sites were considered for the comparison. A non-parametric Passing-Bablok regression analysis using Kendall’s τ as the correlation coefficient revealed a strong positive association between saliva and tissue HPV DNA methylation levels (τ = 0.7483, P < 0.0001). CUSUM test of linearity, maximum value was 8.8165 with an associated P-value of 0.0681, suggesting weak evidence against linearity. Saliva and tissue are quite well calibrated with an estimated slope of 1.000 (95% CI: 0.973, 1.059). Although matching pair examination indicated a mean difference of −0.0181, it was not statistically significant (P = 0.1047) (Figure 3).
Figure 3.
Bland-Altman plot of HPV DNA methylation comparison between match pairs of HNC tumour tissue samples and saliva samples.
HPV DNA methylation changes between HPV-HNC and controls
Considering the differences between HNC and controls in methylation levels in late genes, and considering primer efficiencies, 13 CpG sites located at L1 and L2 genes were selected for further evaluation. A total of 18 HNC saliva samples and 10 control saliva samples were assessed for the methylation levels in the selected sites. From the sites evaluated, statistically significant differences were identified for CpG sites at L2—CpG 6 (P = 0.0004), L1—CpG 3 (P = 0.0144), L1—CpG 2 (P = 0.0395) and L2—CpG 19 (P = 0.0455). L2—CpG 6 was identified as the most differentially methylated site where mean percentage methylation levels were 0.376 and 0.102 for HNC and controls, respectively (Figure 4).
Figure 4.
Comparison of HPV DNA methylation changes in late genes between HPV-HNC and controls. From the sites evaluated statistically significant differences were identified for CpG sites at L2—CpG 6 (P = 0.0004), L1—CpG 3 (P = 0.0144), L1—CpG 2 (P = 0.0395) and L2—CpG 19 (P = 0.0455).
Discussion
Using a custom-designed MALDI-TOF mass spectrometry-based quantitative DNA methylation estimation platform, this study reconfirms that CpG dinucleotides in the HPV genome are susceptible to methylation changes. Due to the absence of intrinsic methyltransferases in HPV, the methylation alterations in HPV DNA are believed to be mediated by the host cellular methylation machinery (11). The purpose of these modifications and their effects are yet to be revealed. However, our findings, highlight that these methylation changes may have a diagnostic potential.
Due to the unavailability of a known precancerous lesion, evaluation of methylation patterns between HPV-HNC and HPV-positive controls requires an alternative platform (17). As HPV DNA can be isolated from the saliva of HPV-HNC patients and HPV-positive controls, we hypothesized that saliva can be used as an intermediate medium to compare the methylation patterns. Supporting our assumption, preliminary data comparing the methylation patterns between tumours and corresponding saliva samples indicated a reasonable concordance. Hence salivary HPV DNA methylation patterns were considered for the subsequent comparisons.
Varying degrees of methylation were detected ranging from no methylation to total methylation in different CpG sites of the considered region including L1, L2 and LCR of HPV16 genome. Despite these variations, relatively higher methylation levels were detected in HPV late genes in the majority of HPV-HNC patients. More importantly, L1 and L2 methylation levels were observed to be lower in control samples compared with the HPV-HNC sample. As such, certain CpG dinucleotides located in L1 and L2 were considered for further evaluation in additional samples considering the differences in methylation in preliminary samples and primer efficiencies. Methylation levels of certain CpG sites including CpG sites at L2—CpG 6 (P = 0.0004), L1—CpG 3 (P = 0.0144), L1—CpG 2 (P = 0.0395) and L2—CpG 19 (P = 0.0455) reached a statistical significance highlighting their diagnostic implications.
Similar findings have been reported by a number of cervical cancer studies. The common observation of these studies is significantly higher methylation levels in L1 and L2 genes in cervical cancer and high-grade cervical pre-cancer compared with low-grade pre-cancer and HPV-positive controls (14,18–21). These observations are not limited to HPV16 but are also shared by many other high-risk HPV types (14,19,20). Even though a comprehensive evaluation of HPV DNA methylation differences between HPV-HNC and controls has been performed previously, several studies have investigated tumour HPV DNA methylation patterns. Using methylated DNA immunoprecipitation sequencing (MeDIP-Seq), Wilson et al. investigated HPV16 DNA methylation patterns in HPV-HNC tissue (N = 3) and reported higher methylation levels at the L1–L2 boundary (22). Similar findings have been reported by Balderas-Loaeza et al. where they detected higher levels of DNA methylation in the HPV L1 region (23). The study conducted by Giuliano et al. also reported higher HPV L1 and L2 methylation levels in oral gargles of HPV-HNC patients (N = 101) (16). The investigators compared these methylation patterns with HPV-positive controls (N = 3) and reported that methylation levels in controls are comparatively lower compared with HPV-HNC (16). Even though higher levels of methylation in viral LCR have been reported by several HPV-HNC studies analysing cell lines and tumour tissue, our preliminary data indicated relatively lower levels of methylation in LCR and hence not evaluated in additional samples. Our findings together with other cervical cancer and HNC studies indicate that higher levels of DNA methylation in HPV late genes are indicative of HPV-HNC risk. As DNA methylation markers are relatively stable in fixed samples and can be easily detected using PCR-based techniques, such biomarkers can be easily adapted for screening purposes and we believe that the incorporation of DNA methylation-based risk stratification can improve the efficacy of salivary HPV-based early detection of HPV-HNC.
Limitations
As this is a preliminary study, the samples numbers are limited. The region containing CpG 9 to CpG 14 in HPV L2 could not be amplified using designed primers. Certain samples with low HPV copy numbers could not be amplified with this method.
Supplementary Material
Acknowledgements
The authors thank Ms. Trang Le, Ms Jennifer Edmunds, Ms Charmaine Micklewright, Ms Jacqui Keller, Ms Dana Middleton and Ms Emma Knowland for the assistance provided with patient recruitment. We would also like to thank Ms Vandhana Bharti from Agena Bioscience, Australia for helping with methylation assay. Graphical abstract was created using image templates from Servier Medical Art (Creative Commons Attribution 3.0 Unported License; https://smart.servier.com).
Glossary
Abbreviations
- HPV
human papillomavirus
- HNC
Head and Neck cancers
- HPV-HNC
HPV-driven HNC
- LCR
long coding region
- SAP
Shrimp alkaline phosphatase
Contributor Information
Chameera Ekanayake Weeramange, Saliva and Liquid Biopsy Translational Laboratory, Griffith Institute for Drug Discovery (GRIDD), Griffith University, Nathan, Queensland 4111, Australia; Menzies Health Institute Queensland (MIHQ), Griffith University, Gold Coast, Queensland 4222, Australia; Faculty of Health, School of Biomedical Science, Centre for Biomedical Technologies, Queensland University of Technology, Kelvin Grove, Queensland 4059, Australia; Department of Medical Laboratory Sciences, Faculty of Health Sciences, The Open University of Sri Lanka, Nugegoda, Sri Lanka.
Kai Dun Tang, EDA School of Biological Sciences and Biotechnology, Nankai International Advanced Research Institute (Shenzhen Futian), Nankai University, Tianjin, 300071, P.R. China.
Darryl Irwin, Agena Bioscience, Bowen Hills, Queensland 4006, Australia.
Gunter Hartel, Statistics Unit, QIMR Berghofer Medical Research Institute, Herston, Queensland 4006, Australia; School of Public Health, The University of Queensland, Brisbane, Queensland, Australia; School of Nursing, Queensland University of Technology, Brisbane, Queensland, Australia.
Julian Langton-Lockton, Metro-North Sexual Health and HIV Service, Brisbane, Queensland 4000, Australia.
Rahul Ladwa, Department of Cancer Care Services, Princess Alexandra Hospital, Woolloongabba, Queensland 4102, Australia; Faculty of Medicine, The University of Queensland, Herston, Queensland 4006, Australia.
Lizbeth Kenny, Faculty of Medicine, The University of Queensland, Herston, Queensland 4006, Australia; Department of Cancer Care Services, Royal Brisbane and Women’s Hospital, Herston, Queensland 4006, Australia.
Touraj Taheri, Faculty of Medicine, The University of Queensland, Herston, Queensland 4006, Australia; Department of Anatomical Pathology, Royal Brisbane and Women’s Hospital, Herston, Queensland 4006, Australia.
Bernard Whitfield, Department of Otolaryngology, Head and Neck Surgery, Princess Alexandra and Logan Hospitals, Meadowbrook, Queensland 4131, Australia; School of Medicine and Dentistry, Griffith University, Gold Coast, Queensland 4222, Australia.
Sarju Vasani, Faculty of Medicine, The University of Queensland, Herston, Queensland 4006, Australia; Department of Otolaryngology, Royal Brisbane and Women’s Hospital, Herston, Queensland 4006, Australia.
Chamindie Punyadeera, Saliva and Liquid Biopsy Translational Laboratory, Griffith Institute for Drug Discovery (GRIDD), Griffith University, Nathan, Queensland 4111, Australia; Menzies Health Institute Queensland (MIHQ), Griffith University, Gold Coast, Queensland 4222, Australia.
Funding
Cancer Australia Grant (APP1145657 to C.P.); National Health and Medical Research Council Grant (APP 2002576 and APP 2012560 to C.P.); the Garnett Passe and Rodney Williams Foundation, NIH R21 and the RBWH Foundation. C.E.W. was supported by a scholarship from the University Grants Commission, Sri Lanka, and Queensland University of Technology, Australia.
Conflict of Interest Statement: None declared.
Data availability
The data underlying this article will be shared on reasonable request to the corresponding author.
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Supplementary Materials
Data Availability Statement
The data underlying this article will be shared on reasonable request to the corresponding author.