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Human papillomavirus detection in dysplastic and malignant oral verrucous lesions


Angela Stokes,1 Eliete Guerra,2 Jon Bible,3 Eugene Halligan,3 Guy Orchard,4 Edward Odell,1 Selvam Thavaraj1
1

Oral Pathology, Department of Clinical and Diagnostic Sciences, Kings College London Dental Institute, London, UK 2 Oral Pathology, Department of Dentistry, Faculty of Health Science, University of Brasilia, Brasilia, Brazil 3 Department of Infection, Virology Section, GSTS Pathology, St Thomas Hospital, London, UK 4 St Johns Institute of Dermatology, GSTS Pathology, St Thomas Hospital, London, UK Correspondence to Dr Selvam Thavaraj, Oral Pathology, Floor 28, Tower Wing, Kings College London Dental Institute, Great Maze Pond, London SE1 9RT, UK; selvam.thavaraj@kcl.ac.uk Accepted 16 November 2011 Published Online First 15 December 2011

ABSTRACT The role of human papillomaviruses (HPV) in dysplastic and malignant oral verrucous lesions is controversial since there is a wide range in the incidence of virus detection. This study used a multi-tiered method of HPV detection using DNA in-situ hybridisation (ISH) for lowand high-risk subtypes, consensus PCR, and HPV genotype analysis in archival tissue from 20 cases of dysplastic and malignant oral verrucous lesions. The biological signicance of HPV DNA detection was assessed by p16 immunohistochemistry (IHC). While 1/7 carcinomas and 5/13 dysplasias contained HPV DNA by consensus PCR and genotype analysis, all specimens were negative for low- and high-risk HPV ISH and negative for p16 IHC. Results show that although highrisk HPV DNA is detectable in a subset of these lesions, the lack of p16 overexpression suggests that the oncogenic process is not driven by HPV oncoproteins.

buccal mucosa, gingiva, lip and oor of mouth. Thirteen cases were non-invasive verrucous or papillary lesions (9 men, 4 women; mean age 69 years, range 57e83) with varying degrees of architectural and/or cytological atypia (gure 1C) lacking any invasive component. Five were from the buccal mucosa, four from the palate, two from the tongue and one each from the gingiva and oor of mouth. Ten tonsils removed for chronic tonsillitis were used as controls.

HPV in-situ hybridisation


HPV DNA ISH was carried out using the Ventana Inform HPV II (low-risk) and III (high-risk) Family 16 Probe (B) on a Benchmark Ultra Autostainer (Ventana Medical Systems, Cambridgeshire, United Kingdom) and scored as previously described.7 Laryngeal papillomatosis, CaSki, HeLa and C-33A cells served as controls.

INTRODUCTION
The clinical and histological appearances of premalignant and malignant verrucous lesions of the oral cavity have led some investigators to examine the association between human papillomaviruses (HPV) and this group of lesions. The reported incidence of HPV in oral verrucous carcinoma (VC) ranges from 0% to 100%.1e4 This inconsistency may be due to the lack of standardised detection methods and no absolute histological dening criteria for verrucous dysplasia or carcinoma. This study aimed to determine whether high- or low-risk subtypes of HPV are present in dysplastic or malignant oral squamous verrucous lesions using DNA in-situ hybridisation (ISH), consensus PCR and HPV genotype analysis by multiplex PCR. The biological signicance of detectable HPV DNA was assessed by p16 immunohistochemistry, a surrogate marker for viral oncoprotein expression.

Detection of HPV DNA by PCR


DNA was extracted from 25 mm sections of FFPE biopsies using the QIAamp DNA Tissue kit (Qiagen, Crawley, UK) following standard operating procedures. Ampliable DNA was veried by b-globin gene PCR, then HPV DNA was analysed by PCR using GP5+/6+ primers to detect low- and high-risk genotypes.8 DNA from CaSki cells served as a positive control and DNA from UPCI:SCC089 cells and omission of template DNA served as negative controls.

HPV genotype analysis


Samples were genotyped using a proprietary kit (digene LQ Test RUO, Qiagen) utilising a multiplex bead-based xMAP assay according to the manufacturers instructions as previously described.7 Positive and negative controls were as for PCR (above).

p16 Immunohistochemistry
p16 Immunohistochemistry (IHC) was carried out using a proprietary kit (CINtec Histology, mtm Laboratories, Heidelberg, Germany) as previously described.7 Tonsil carcinoma with p16 overexpression and omission of primary antibody served as positive and negative controls, respectively.

MATERIALS AND METHODS Patients and samples


Formalin-xed parafn-embedded samples (FFPE) from 20 patients demonstrating dysplasia or carcinoma with verrucous or papillomatous morphology were included in this study. Of these, seven were carcinomas (5 men, 2 women; mean age 76 years, range 58e85) that either fullled the criteria of VC as previously described5 6 or demonstrated conventional well differentiated squamous cell carcinoma with an exophytic verrucous or papillary architecture (gure 1A,B). Three were from the tongue and one each from
J Clin Pathol 2012;65:283e286. doi:10.1136/jclinpath-2011-200454

RESULTS
Results are summarised in table 1.

HPV DNA ISH, consensus HPV PCR and HPV genotype analysis
All samples were negative for low- and high-risk HPV by ISH. HPV DNA was detected in 1 of 7
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Short report

Figure 1 Histological appearance of oral verrucous lesions. (A) Representative photomicrograph of an invasive verrucous lesion (original magnication 320). (B) Representative photomicrograph of a dysplastic papillary lesion (original magnication 340). (C) All lesions showed architectural and cytological atypia to varying degrees (original magnication 3200). carcinomas and 5 of 13 dysplasias by GP5+/6+ PCR, all of which were positive for HPV16 by genotype analysis. All cases of chronic tonsillitis were negative for HPV by GP5+/6+ PCR. Table 1
Lesion Carcinoma Carcinoma Carcinoma Carcinoma Carcinoma Carcinoma Carcinoma Dysplasia Dysplasia Dysplasia Dysplasia Dysplasia Dysplasia Dysplasia Dysplasia Dysplasia Dysplasia Dysplasia Dysplasia Dysplasia

p16 Immunohistochemistry
Most samples showed no nuclear or cytoplasmic reactivity for p16 IHC. However, 1 of 7 carcinomas and 4 of 13 dysplasias

Summary of human papillomavirus (HPV) DNA detection and p16 immunohistochemistry results
Site Alveolar ridge Tongue Tongue Lip Floor of mouth Buccal mucosa Tongue Buccal mucosa Hard palate Alveolar ridge Hard palate Hard palate Hard palate Buccal mucosa Buccal mucosa Tongue Buccal mucosa Floor of mouth Tongue Buccal mucosa Low-risk HPV, ISH e e e e e e e e e e e e e e e e e e e e High-risk HPV, ISH e e e e e e e e e e e e e e e e e e e e Consensus primer PCR e e e e e e + + e + e e e + + e e e e + Genotype e e e e e e 16 16 e 16 e e e 16 16 e e e e 16 p16 e e e e e e* e e* e e e e e e* e* e e* e e e

*Denotes patchy and/or focal p16 staining insufcient to meet the criteria of HPV oncoprotein driven p16 overexpression.

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J Clin Pathol 2012;65:283e286. doi:10.1136/jclinpath-2011-200454

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Short report
showed variable nuclear and/or cytoplasmic staining limited to the basal and suprabasal compartments (gure 2A). One case of carcinoma showed clear demarcation of the lesion from adjacent normal epithelium (gure 2B). None of the cases fullled the criteria of HPV oncoprotein-mediated p16 overexpression.9 areas of verrucous leukoplakia.6 This variation in diagnostic approach may explain the lack of consensus on the association between this group of lesions and HPV. In order to circumvent this lack of standardisation of diagnostic criteria, we have chosen not to distinguish between verrucous hyperplasia and dysplasia, nor between VC and conventional squamous cell carcinoma with verrucous/papillary morphology. The current and previous studies are limited by small sample sizes due to the rarity of this group of lesions. Nevertheless, the lack of consensus on the role of HPV in these lesions is likely to be partly due to the diverse sensitivity and specicity of the different detection methods. Most studies investigating the association between oral verrucous lesions and HPV to date have employed a single detection step,3 4 raising the possibility of false positive results. In this study, we used multiple detection methods: DNA in-situ hybridisation and PCR for the consensus L1 region. This approach combined the high specicity of the former technique with the high sensitivity of the latter.7 11 Our data showed that although HPV DNA was detectable by PCR, the negative ISH suggests that viral DNA was present in very low copy numbers. The presence of HPV DNA should be interpreted with caution because the biological signicance of detectable viral DNA is unknown. In the current study, no viral DNA was detected in the control group. Furthermore, others have shown no signicant difference in the incidence of HPV between VC and controls,12 suggesting that any detectable HPV is likely to be due to transient infection. We tested the biological signicance of detectable HPV in our cohort using p16 IHC. Only strong diffuse nuclear and cytoplasmic staining homogeneously distributed throughout the lesion is indicative of oncoprotein-driven p16 overexpression9; all other staining patterns are likely due to undened mechanisms of p16 overexpression. One case in our series demonstrated strong p16 expression limited to basal and suprabasal lesional epithelium with abrupt transition to normal epithelium. However, HPV DNA was not detectable in this case, further supporting the likelihood that p16 overexpression arose by HPV-independent mechanisms. While it may be possible for these lesions to arise through p16-independent mechanisms, previous studies have shown that low-risk HPV do not occur in isolation without co-infection with high-risk types.12 Therefore, any persistent HPV infection is likely to result in p16 overexpression.

DISCUSSION
The frequent association between HPV and ano-genital VCs, a supercial similarity to papillomas and condylomas, and the observation of koilocyte-like prickle cells, have led several investigators to examine the possibility of an association between HPV and dysplastic and malignant oral squamous verrucous or papillary lesions. However, the terms verrucous hyperplasia and verrucous dysplasia both lack absolute morphological dening criteria. This is further confounded by the rarity of these lesions and the fact that several authorities consider verrucous hyperplasia to be premalignant.2 10 The situation is somewhat similar with carcinomas displaying verrucous morphology since diagnostic criteria are applied with variable stringency in the diagnosis of VC. Furthermore, some conventional well differentiated squamous cell carcinomas may contain papillary foci or arise in

CONCLUSION
We have shown that while high-risk HPV DNA appears to be detectable in a subset of malignant and dysplastic oral verrucous lesions, the lack of p16 overexpression suggests that the

Take home messages


< The incidence of HPV detection in oral verrucous lesions

varies widely.
< This study used a multi-tiered method of HPV detection: DNA

Figure 2 p16 Immunohistochemistry. (A) Variable p16 expression that did not full the criteria of human papillomavirus (HPV) oncoproteindriven p16 overexpression (original magnication 320). (B) Strong diffuse basal and suprabasal p16 staining with clear demarcation between the verrucous lesion and adjacent non-dysplastic epithelium in a case that was negative for HPV DNA (original magnication 320).
J Clin Pathol 2012;65:283e286. doi:10.1136/jclinpath-2011-200454

in-situ hybridisation, PCR for the consensus L1 region, genotype analysis by multiplex PCR and p16 IHC. < Although HPV DNA was detected in a subset of oral verrucous lesions, the lack of associated p16 over-expression suggests that the neoplastic process may not be driven by high-risk HPV.

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Short report
REFERENCES

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oncogenic process may not be driven by high-risk HPV. This study is limited by the small cohort size of this uncommon group of entities. Further work is required to ascertain whether the virus is transient or persistent, to determine its biological signicance and to elucidate non-HPV causative factors.
Competing interests None. Ethics approval No specic ethics approval required under Guys & St Thomas NHS Foundation R&D Department (RJ110/N200). Contributors All authors contributed signicantly to the manuscript. Provenance and peer review Not commissioned; externally peer reviewed.

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Human papillomavirus detection in dysplastic and malignant oral verrucous lesions


Angela Stokes, Eliete Guerra, Jon Bible, et al. J Clin Pathol 2012 65: 283-286 originally published online December 15, 2011

doi: 10.1136/jclinpath-2011-200454

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