O R I G I N A L A R T I C L E
The role of human papillomavirus in p16-positive oral cancers
Simone Belobrov
1| Alyssa M. Cornall
2,3,4| Richard J. Young
5| Kendrick Koo
6| Christopher Angel
7| David Wiesenfeld
1,6,8| Danny Rischin
9| Suzanne M. Garland
2,3,4| Michael McCullough
11Melbourne Dental School, Faculty of Medicine Dentistry and Health Science, The University of Melbourne, Melbourne, VIC, Australia
2Regional HPV Labnet Reference
Laboratory, Department of Microbiology and Infectious Diseases, The Royal Women’s Hospital, Melbourne, VIC, Australia
3Murdoch Childrens Research Institute, Melbourne, VIC, Australia
4Department of Obstetrics and
Gynaecology, The University of Melbourne, Melbourne, VIC, Australia
5Research Division, Peter MacCallum Cancer Centre, Melbourne, VIC, Australia
6Department of Surgery, The Royal Melbourne Hospital, The University of Melbourne, Melbourne, VIC, Australia
7Department of Anatomical Pathology, Peter MacCallum Cancer Centre, Melbourne, VIC, Australia
8Head and Neck Oncology Tumour Stream, Victorian Comprehensive Cancer Centre, Melbourne, VIC, Australia
9Divison of Cancer Medicine, Peter MacCallum Cancer Centre and The University of Melbourne, Melbourne, VIC, Australia
Correspondence
Michael McCullough, Melbourne Dental School, The University of Melbourne, Melbourne, VIC, Australia.
Email: m.mccullough@unimelb.edu.au
Funding information
The Melbourne Dental School, The University of Melbourne; Australian Dental Research Foundation; Price Family Foundation; Australian Government Research Training Program Scholarship
Background: The aim of this study was to identify the presence and frequency of human papillomavirus (HPV) nucleic acid in p16-positive oral squamous cell carcino- mas (OSCCs), to assess whether the virus was transcriptionally active and to assess the utility of p16 overexpression as a surrogate marker for HPV in OSCC.
Methods: Forty-six OSCC patients treated between 2007 and 2011 with available formalin-fixed paraffin-embedded (FFPE) specimens were included. Twenty-three patients were positive for p16 by immunohistochemistry (IHC) and these were matched with 23 patients with p16-negative tumours. Laser capture microdissection of the FFPE OSCC tissues was undertaken to isolate invasive tumour tissue. DNA was extracted and tested for high-risk HPV types using a PCR-ELISA method based on the L1 SPF10 consensus primers, and a real-time PCR method targeting HPV-16 and HPV-18 E6 region. Genotyping of HPV-positive cases was performed using a reverse line blot hybridization assay (Inno-LiPA). RNAScope
â(a chromogenic RNA in situ hybridization assay) was utilized to detect E6/E7 mRNA of known high-risk HPV types for detection of transcriptionally active virus.
Results: HPV DNA was found in 3 OSCC cases, all of which were p16 IHC-positive.
Two cases were genotyped as HPV-16 and one as HPV-33. Only one of the HPV- 16 cases was confirmed to harbour transcriptionally active virus via HPV RNA ISH.
Conclusion: We have shown that the presence of transcriptionally active HPV rarely occurs in OSCC and that p16 is not an appropriate surrogate marker for HPV in OSCC cases. We propose that non-viral mechanisms are responsible for the majority of IHC p16 overexpression in OSCC.
K E Y W O R D S
head and neck cancer, human papilloma virus, oral cancer, oral squamous cell carcinoma, p16
1 | I N T R O D U C T I O N
Head and neck squamous cell carcinomas (HNSCC) are the sixth most common cancer worldwide.1Tobacco and alcohol use are the
principal risk factors. However, 25%-35% have been shown to be associated with human papillomavirus (HPV), and these are mostly confined to the tonsil and base of tongue.2,3 Patients with HNSCC HPV-positive oropharyngeal tumours have distinct clinical features
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and a more favourable prognosis3 compared to HPV-negative HNSCC.
The oral cavity is the most common non-oropharyngeal HNSCC site where HPV is implicated.4Nevertheless, the role of HPV in oral cancer is controversial, with reported prevalence of HPV ranging from 0% to 90%.2,5-7Some studies may have mixed oral cavity and base of tongue tumours, leading to falsely higher rates of HPV in oral cancer. At present, the prevalence and role of HPV in non-oro- pharyngeal sites remains unclear and a causal relationship has not been established. Immunohistochemical (IHC) staining for p16 is used as a surrogate marker for HPV infection in oropharyngeal SCC, with HPV-positive tumours overexpressing p16.8
HPV oncogenes E6 and E7 are required for tumour initiation and immortalization.9 They are highly associated with carcinogenetic activity and are responsible for inhibition of the tumour suppressors p53 and retinoblastoma protein (pRb).10,11Moreover, E7-driven inac- tivation of pRb in high-risk (HR) HPVs has been shown to lead to p16 overexpression.3,12 This interferes with cell cycle control and promotes genetic instability and cancer progression.11,13
In a previous study,14we performed retrospective immunohisto- chemical (IHC) staining to determine the expression of several pro- teins in 129 formalin-fixed paraffin-embedded (FFPE) specimens from a well-defined cohort of consecutive oral squamous cell carci- noma (OSCC) patients. We found 23 (18%) of these patients dis- played p16 overexpression, which was significantly increased in non- smokers and non-drinkers who were less than 70 years old. This result warranted further investigation given the association between p16 overexpression and HPV in HNSCC. We hypothesize that if p16 could be used as a surrogate marker for HPV in OSCC, it may fur- ther indicate prognosis.
Our first aim for this study was to determine whether p16 IHC- positive OSCCs actually harbour HPV. Secondly, we aimed to iden- tify either HPV E6 or E7 mRNA in these tumours as a marker of transcriptionally active virus that may be responsible for oncogenesis in these patients. Finally, we aimed to assess the utility of p16 as a surrogate marker for HPV infection in OSCC.
2 | M A T E R I A L S A N D M E T H O D S
2.1 | Patients
Twenty-three OSCC p16 IHC-positive patients were matched to a control cohort of 23 OSCC p16 IHC-negative patients. Matching was performed using semi-parametric and nonparametric matching meth- ods as implemented in the MatchIt R package.15 Matching was attempted on age, gender, T stage (divided into early [T1-T2] and late [T3-T4] stages), N stage (divided into node-positive and node-nega- tive), tobacco use (which included those with >5 pack years) and alco- hol use (which included <3 drinks per week). Exact matches were found for gender, T stage, N stage and tobacco use, with a similar mean age between the two groups. Archived FFPE sections were obtained for each of the 46 patients. All tumours were in oral cavity sites only. Mortality data were sourced from both the ACCORD (the
Australian Comprehensive Cancer Outcomes and Research Database) and the Victorian Cancer Registry databases (census date 31 Decem- ber 2015). Expression levels of p16 were determined by IHC staining using p16 mouse monoclonal antibody (Roche, Basel, Switzerland).
The staining was scored as previously described.14
This study was approved by the Melbourne Health Human Research Ethics Committee 19 September 2012 (MH Project num- ber 2012.071).
2.2 | Histological preparation of samples
Histological sectioning of FFPE tissue blocks was performed by con- ventional sterile technique at the laboratory service of the Anatomi- cal Pathology Department, within the Royal Melbourne Hospital, Melbourne, Australia. To minimize cross-contamination between sec- tioning of each tissue sample, the microtome blade was changed and the microtome surface cleaned with xylene. Each section was mounted on superfrost plus slides. Haematoxylin and eosin (H&E)- stained sections were reviewed and annotated by a consultant pathologist to define tumour-containing regions.
2.3 | Laser capture microdissection (LCM) and HPV DNA Detection
HPV DNA detection was performed in the Western Pacific Regional HPV Labnet Reference Laboratory within the Molecular Microbiology Department of the Royal Women’s Hospital, Melbourne, Australia; 4- lm-thick mounted FFPE sections were deparaffinized via sequential 5-minute incubations in 100% xylene (twice) and 100% ethanol (twice). The slides were then incubated at 37°C degrees for one hour.
Following this, the annotated H&E-stained tissue section was used to guide capture of the tumour area of interest on the Veritas 704 Laser Capture Microdissecting System (Arcturus Bioscience, Mountain View, CA, USA). Cells were captured onto a thermoplastic CapSure Macro LCM Cap (Applied Biosystems, Foster City, CA, USA) over the target tissue by pulsing with an infrared laser to adhere the cells to the cap.
DNA was extracted from each cap with the Arcturus PicoPure DNA Extraction Kit (Applied Biosystems) as per protocol F of the PicoPure DNA Extraction Kit user guide. DNA from LCM-extracted tissue sec- tions was first tested for extraction efficiency by assessing the pres- ence of intact endogenous human genomic DNA. This was established by performing a 110-base pair (bp) beta-globin real-time PCR assay using the LightCyclerâ 480 Instrument II (Roche) as previously described.16All PCR conditions are outlined in Table 1.
LCM-extracted DNA was then tested for HPV DNA on the DNA ELISA kit HPV SPF10, V.1 (Labo Bio-medical Products BV, Rijswijk, the Netherlands), which is validated for detection of 44 HPV geno- types, according to the manufacturer’s instructions. Samples that were positive for HPV DNA on the DNA ELISA were further tested for HPV genotype on the RHA kit HPV SPF10-LiPA25, V.1 (Labo Bio-medical Products BV). Hybridization of the biotinylated ampli- cons to the detection strip was performed using the Auto-LiPA 30 instrument (Innogenetics) according to the manufacturer’s
instructions.17 SPF10-LiPA allows for identification of 25 different HPV genotypes, which include 15 high-risk types (HPV-16, 18, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68 and 73) and 8 low-risk types (HPV-6, 11, 34, 40, 43, 44, 54, 70 and 74). Additional screen- ing of all samples for HPV-16 and HPV-18 E6 gene via real-time PCR assay was performed using the LightCyclerâ480 Instrument II (Roche) as previously described,16 targeting a 102-bp fragment of the HPV-16 E6 and a 142-bp fragment of the HPV-18 E6 regions.
2.4 | HPV RNA detection
RNA in situ hybridization (ISH) for detection of high-risk HPV E6/E7 mRNA was performed in the Translational Research Laboratory within the Peter MacCallum Cancer Centre of the Victorian Comprehensive Cancer Centre, Melbourne, Australia, using the RNAScopeâHPV kit (Advanced Cell Diagnostics, Hayward, CA, USA) according to the man- ufacturer’s instructions. RNA ISH was performed on all cases in which HPV DNA was detected by PCR (n= 3) to assess for the presence of transcriptionally active virus, and also as an extra test on all cases where the 110-bp beta-globin was not detected (n= 10). One FFPE section from each case was hybridized with a single cocktail target HRP-labelled probe of 18 high-risk (HR) HPV genotypes (16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, 73 and 82). A second FFPE section from each case was used as a positive control and hybri- dized with probes to endogenous peptidyl-prolyl cis-trans isomerase B (PPIB) mRNA, to demonstrate detectible mRNA in the FFPE samples.
Specific staining signals when present were identified as brown, punc- tate dots located in the cytoplasm and/or nucleus and qualitatively classified in a binary manner as either positive (present) or negative (absent). Only cases showing positive control probe signals were scored for the presence or absence of HPV probe signals.
Following RNAScopeâISH, seven cases remained inconclusive as testing did not detect either of the internal controls, 110-bp beta- globin DNA target on LCM-extracted DNA or PPIB mRNA target on ISH. Subsequently, a shorter fragment (70 bp) human genomic beta- globin qPCR was designed and validated (validation data not shown).
The assay comprised of PC03_4 TaqMan probe and PCO3 forward primer, with a new reverse primer, PCO5 (50- GTGCATCTGACTCCT GAGGA-30). The 70-bp beta-globin qPCR was performed on the LCM-extracted DNA from all inconclusive cases to confirm the pres- ence of endogenous human genomic DNA.
2.5 | Statistical analysis
Statistical analysis was performed using the R software package (R Foundation for Statistical Computing, Vienna, Austria). The 5-year
survival rate was analysed using the Kaplan-Meier method. Hazard ratios were obtained using the univariate Cox proportional hazards model. Statistical significance was set at P≤ .05.
3 | R E S U L T S
The study population consisted of 23 patients with p16-positive OSCC and a further 23 matched patients with p16-negative OSCC.
Of the 46 patients, 33 (72%) were under 70 years of age and were mainly smokers (32 patients, 70%) or alcohol drinkers (29 patients, 63%). Tumour sites were spread across the oral cavity, with the highest proportion (43%) located in the oral tongue (anterior two- thirds of the tongue). Thirty per cent of cancers were stage I. At 5 years, 28% of patients had died from their disease (Table 2). p16 expression was not found to be associated with 5-year disease-spe- cific survival (P= .694 by log rank test) (Figure 1). However, p16- positive patients were found to have a lower risk of death from OSCC than p16-negative patients (HR= 0.47; 95% CI, 0.14-1.55).
HPV L1 DNA was found by SPF10-LiPA PCR assay in 3 male smoking and drinking patients who were all p16-positive. All 3 patients were alive at the census date. Genotyping identified 2 cases as HPV-16 and the remaining case as HPV-33. The HPV-33 and one of the HPV-16 cases were strongly positive by SPF10-LiPA assay with the optical density of their PCR products being four times greater than the HPV-positive cut-off value on ELISA. This resulted in a dark hybridization band on LiPA. The other HPV-16 case was borderline positive, with the optical density of the PCR product at the HPV-positive cut-off value on ELISA resulting in a lighter band on LiPA (Figure 2). HPV-16 E6 DNA was also identified in the case that was strongly positive for HPV-16 L1 DNA. All other samples were HPV-16/HPV-18 E6 DNA-negative. Housekeeping genes could not be detected in 2 of the 46 samples and these were excluded from further analysis, as they could not definitively be considered as HPV-negative.
RNAScopeâISH was performed for the 13 samples which were either HPV DNA-positive by PCR (as a confirmatory test for tran- scriptionally active virus) or 110-bp beta-globin-negative (as an alter- native test). Eight of these samples were p16-positive and 5 were p16-negative. Housekeeping RNA was detected in 6 samples, but was most likely degraded in the 7 samples in which the housekeep- ing/control mRNA probe was undetectable, including the HPV-33- positive sample. Only 1 of the 3 HPV DNA PCR-positive samples (which was both HPV-16 L1- and HPV-16 E6-positive by PCR) was also positive for the high-risk HPV mRNA RNAScopeâ ISH probe (Figure 3). This mRNA was not identified in the other HPV-16 L1- T A B L E 1 Thermocycler conditions for each PCR target
Target Initial denaturation Amplification Number of cycles Cooling
Beta-globin 95°C for 5 min 95°C for 10 s, 60°C for 30 s, 72°C for 1 s 45 40°C for 1 min L1-SPF10 94°C for 9 min 95°C for 30 s, 52°C for 45 s, 72°C for 45 s 40 72°C for 5 min HPV-16/18 E6 95°C for 10 min 95°C for 10 s, 55°C for 10 s, 65°C for 20 s 55 40°C for 1 min
positive sample. HPV DNA or mRNA was not detected in any of the p16-negative samples (Table 3).
In summary, of the 44 cases, only one sample was HPV E6/E7 mRNA-positive and three samples were HPV DNA-positive. Within the study period, twelve patients had died of disease and three from other causes. Too few HPV-positive tumours were identified to per- form meaningful statistical analysis.
4 | D I S C U S S I O N
Most studies assessing the role of HPV in OSCC utilize DNA detection as an indicator of the presence of HPV. This is the first study, to our
knowledge, to examine OSCC for evidence of HPV by two orthogonal methods in archived FFPE tissues: tumour-associated DNA using LCM-PCR and transcriptional activity by mRNA ISH. Using both HPV DNA and RNA detection methods, we have shown that HPV may be a causative factor in OSCC, albeit at low prevalence (1 patient, 2.3%) in our small cohort. Our frequency of HPV-positive tumours, using more precise laboratory methods, is concordant with the existing litera- ture.13,18We have shown that p16 overexpression is only rarely HPV- related and p16 IHC expression cannot be used as a surrogate marker for the presence of HPV in oral cavity carcinomas.
It was surprising that so few samples were HPV-positive, with just 3 of 44 cases containing detectable tumour-associated HPV DNA. Two cases harboured HPV-16, and a further case harboured HPV-33. Stud- ies have shown that HPV-16 is the most frequent viral subtype found in HNSCC, followed by HPV-33.19However, detection of HPV DNA does not in itself correlate with biological activity and oncogenesis,20 especially in the absence of E6/E7 transcripts.21Only one HPV DNA- positive OSCC in our cohort was also positive for HPV E6/E7 mRNA, genotyped as HPV-16. This is the only case in our cohort we can confi- dently attribute to HPV. The SPF10-LiPA PCR result in the other HPV- 16-positive sample was borderline positive, and this sample was also negative by HPV-16/HPV-18 E6 PCR and RNA ISH. This was not unex- pected as the SPF10-LiPA assay has a higher sensitivity due to the shorter amplicons (65 bp) covering the target. This cancer cannot con- clusively be considered as HPV-driven, with no evidence of viral tran- scription. The mRNA control was not detected in the HPV-33 sample, rendering this case inconclusive for transcriptionally active infection.
There is no consensus method to detect HPV in HNSCC, espe- cially for archived FFPE tissues. HPV identification in FFPE tissues is challenging, as both DNA and RNA are degraded by formalin fixa- tion, age and variable storage conditions, which reduces PCR effi- ciency and assay sensitivity.22The analysis of RNA in this archival tissue is particularly challenging as tissue autolysis prior to fixation can result in degradation of RNA of resected tumours, and RNA is T A B L E 2 Clinicopathological features of our patient cohort
p16 IHC- positive
p16 IHC-
negative Total % Gender
Male 13 13 26 57
Female 10 10 20 43
Age
<70 17 16 33 72
>70 6 7 13 28
Tobacco
Smoker 16 16 32 70
Non-smoker 7 7 14 30
Alcohol
Drinker 13 16 29 63
Non-drinker 10 7 17 37
Stage
I 6 8 14 30
II 6 5 11 24
III 4 5 9 20
IV 7 5 12 26
Site
Tongue (anterior one-third) 12 8 20 43
Mandibular Alveolus 1 1 2 4
Maxillary Alveolus 2 5 7 15
Floor of Mouth 1 4 5 11
Hard Palate 1 1 2 4
Cheek Mucosa 3 2 5 11
Vestibule of Mouth 0 0 0 0
Retromolar Region 3 2 5 11
Outcome after 5 years
Deceased 5 8 13 28
Alive 18 15 33 72
Non-smoking status has been defined as a negligible history of tobacco use and non-drinking status as no regular alcohol consumption or previ- ous history of heavy alcohol intake or abuse. Stage has been classified according the American Joint Committee on Cancer (AJCC) sixth edition system.
F I G U R E 1 Kaplan-Meier survival curve comparing p16-positive and p16-negative populations
cross-linked to adjacent molecules (DNA and proteins) during forma- lin fixation23and can be further damaged by prolonged storage time and poor storage conditions of paraffin blocks.24
The utilization of SPF10 primers for the DNA analysis has been shown to be ideal and have a higher sensitivity for degraded HPV sequences given their shorter amplicon lengths.17,25In FFPE, this is superior to other clinical assays available26that broadly target the L1 region17and enable the amplification of multiple HPV types.27SPF10 HPV PCR can be combined with a reverse hybridization line probe assay (LiPA) permitting simultaneous detection of different HPV types individually in a single assay.25Additional RT-PCR assays with primers targeting the HPV-16 and HPV-18 E6 region are necessary as HPV L1 regions can be lost following HPV DNA integration into the host gen- ome, leading to false negatives in more advanced disease.4 The E6 nucleotide sequence exhibits less nucleotide variation,28and targeting two different viral gene locations improves sensitivity.
RNA detection was achieved by RNA ISH (RNAScopeâ) targeting the actively transcribed E6/E7 mRNA region to verify viral integra- tion and subsequent mRNA transcription. This assay would ideally have been performed on the entire patient cohort. However, in pre- liminary attempts to optimize the assay, the housekeeping/control mRNA probe could not be detected in 20 of 30 cases. This is likely as a result of RNA degradation in our samples, which is unavoidable in FFPE tissues that have been stored for some time. Further analy- sis was limited by the amount of FFPE material available.
The prognostic significance of p16 IHC overexpression has been attributed to improved outcome in oropharyngeal cancer (OPC) cases.29p16 overexpression has been shown to correlate with HPV infection in malignant oral lesions30 with some authors advocating its use as a surrogate marker. Accurate identification of HPV-driven OPC is still a major issue. Algorithms combining p16 and HPV DNA testing perform better than p16 testing alone in OPC cases31 and we hypothesized that the same might be true for non-OPC HNSCC such as OSCC. However, we found that whilst all patients in the cohort with detectable HPV were p16 IHC-positive, p16 expression was not a predictor of HPV status in OSCC. We also found no sta- tistically significant association between p16 overexpression and sur- vival in our study cohort, which is similar to studies involving non- OPC patient cohorts,18,32 but is in contrast to the study by Chung et al.33
Whilst our results have shown that there is no proven prognostic difference based on HPV status in this small cohort of OSCC, F I G U R E 2 INNO-LiPA human papillomavirus (HPV) Genotyping
extra nitrocellulose membrane strips in lanes 1-5. Hybridization bands are analysed by comparing the probe number to the standard grid provided by the manufacturer. Combinations of single or multiple probes correspond to specific HPV types. Lane 1 is the positive control, lane 2 is the negative control, and lanes 3-5 are HPV L1 DNA-positive samples
(A) (B)
F I G U R E 3 A, RNAScopeâdetection of E6/E7 mRNA transcripts as identified as punctate, brown nuclear staining in tumour cells (arrows showing some examples of nuclear signals). B, p16-positive immunohistochemistry staining.
Magnification9400
additional HPV-specific tests could be implemented in OSCC cases pending a large-scale cost/benefit analysis. It is hard to justify the implementation of a screening programme from results in the pre- sent study on the basis that only one case harboured transcription- ally active HPV. However, if this were to occur, we would recommend that LCM be performed prior to sensitive PCR-based HPV testing, to reduce false-positive results. The oral cavity can be colonized by oncogenic HPV without disease, usually cleared within the first year,34and PCR may detect HPV infection in normal epithe- lium in addition to tumour tissue.35 LCM prior to DNA extraction precisely selects tumour cells and prevents contamination by adja- cent tissue.36LCM-PCR has been shown to be the preferred method for HPV detection in cervical neoplasms.37 Further analysis should be performed in positive samples by RNAScopeâISH to confirm the presence transcriptionally active HPV. This has a potential laboratory turn-around time of 2-3 days.
Recent epidemiological data reported 300 400 new cancers of oral cavity and lip cancers in 2012 globally, which resulted in 145 400 deaths.38 For over 50% of cases diagnosed annually, oral cancer remains a lethal disease.1 Treatment might be person- alized for the small subgroup of patients with HPV-positive OSCC, with the development of less morbid treatment regimens.
From a public health perspective, there are further benefits to a HPV vaccination programme, reducing the prevalence of yet another cancer.
The role of HPV in OSCC is more nuanced than in the orophar- ynx, with our study suggesting a non-viral cause of p16 overexpres- sion in this group. We do not recommend that p16 IHC alone be used as a surrogate marker for the presence of HPV in OSCC.
A C K N O W L E D G E M E N T S
The authors acknowledge the financial support from The Melbourne Dental School, The University of Melbourne, the Australian Dental Research Foundation, the Price Family Foundation and the Aus- tralian Government Research Training Program Scholarship. Addi- tionally, we would like to acknowledge BioGrid for assistance with the ACCORD Head and Neck Database.
C O N F L I C T O F I N T E R E S T None declared.
O R C I D
Simone Belobrov http://orcid.org/0000-0001-7103-9410
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T A B L E 3 Summary of results from the various methods used to analyse samples
DNA control (n= 46)
HPV DNA PCR (n= 46)
RNA ISH PPIB Control (n= 13)
HPV mRNA ISH (n= 13)
Positive Negative Positive Negative Positive Negative Positive Negative p16 IHC-
positive
23 0 3 20 6 2 1 7
p16 IHC- negative
21 2 0 23 0 5 0 5
Total 44 2 3 43 6 7 1 12
“DNA control” includes both 70-bp and 110-bp beta-globin PCR housekeeping gene; “HPV DNA”
includes both SPF10-Lipa PCR and HPV-16/18 E6 qPCR. Only one RNA ISH result was mRNA HPV-positive, which was positive to the PPIB control and p16 IHC-positive.
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How to cite this article: Belobrov S, Cornall AM, Young RJ, et al. The role of human papillomavirus in p16-positive oral cancers. J Oral Pathol Med. 2018;47:18–24.
https://doi.org/10.1111/jop.12649
