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J Clin Pathol. 2007 April; 60(4): 439–441.
PMCID: PMC2001116

Human papilloma virus in squamous carcinoma of the head and neck: a study of cases in south east Scotland

Abstract

Several studies have found human papillomavirus virus (HPV) in tissue from head and neck squamous cell carcinomas (HNSCCs), although the number of positive cases varies greatly from study to study. The extent and molecular epidemiology of HPV in HNSCC were assessed within cases drawn from southeast Scotland by performing broad‐spectrum, real‐time HPV polymerase chain reaction (PCR) on DNA extracted from 100 cases of HNSCC in formalin‐fixed, paraffin wax‐embedded material. All HPV‐positive specimens were genotyped and sampled by laser capture microdissection. Pure samples of tumour, and, where possible, dysplastic and normal epithelium were then submitted for further HPV PCR and genotyping to investigate the sensitivity of the technique in small tissue samples. 10 of 100 cases tested positive for HPV, with 8 of these being derived from Waldeyer's ring. HPV DNA was found in adjacent epithelium in two of four cases where this was available. These findings confirm that HPV is likely to be involved in a subset of HNSCC in this population and that successful amplification of HPV nucleic acid is possible even using small amounts of paraffin wax‐embedded tissue.

Involvement of persistent human papilloma virus (HPV) infection in the development of squamous carcinoma of the cervix is now established. Accumulated data have also shown the presence of HPV DNA in head and neck squamous cell carcinomas (HNSCCs), suggesting a similar aetiological role for HPV in these tumours. Specifically, several studies have implicated the oropharynx and, in particular, Waldeyer's tonsillar ring as a predilection site for HPV‐associated carcinogenesis,1,2,3 and have suggested that these tumours represent a distinct clinical subgroup of squamous carcinomas with an improved prognosis.4 This evidence has led to suggestions that HPV DNA detection might be used in the clinical management of HNSCC5 provided that the detection technology was sufficiently robust for preserved pathological specimens.

Materials and methods

Case selection

In all, 101 archival cases of HNSCC were selected, composed of 25 women and 76 men aged 21–87 years (mean age 61.45 years). The cases included 64 laryngeal carcinomas (mean (SD) age 62.63 (10.73) years) and 36 carcinomas from around Waldeyer's ring (tonsil, base of tongue, posterior pharynx and uvula; mean (SD) age 61.06 (11.19) years). A total of 24 controls of benign tonsillar tissue were also identified, comprising 14 females and 10 males aged 14–87 (mean 35.1) years.

DNA extraction and PCR protocol

Thick sections were cut from each paraffin wax block and DNA was extracted using lysis buffer containing proteinase K at 50°C for 4 h. Real‐time PCR incorporating HPV‐specific broad‐spectrum primers was performed. Full details of the methods are as described by Cubie et al.6 A 150 nucleotide product was amplified using the GP5+6+ primers7 on a LightCycler. A positive HPV product was identified via Tm of generated amplicon visualised by incorporation of Sybr Green dye during amplification. Any positive sample identified by the real‐time protocol was subjected to the Roche line blot assay for genotyping, which involved amplification using the PGMY primer set8 designed to generate a 450 nucleotide amplicon. Control primers for the amplification of the β globin gene were also included in the PCR to assess the integrity of the cellular extract. Amplicons were then hybridised to a nylon strip containing 27 immobilised type‐specific HPV probes and 2 levels of β globin control probes according to the technique described by Gravitt et al.9

Laser capture microdissection

Each tumour that tested positive for HPV DNA was microdissected using laser capture microscopy. Sections of thickness, 7 µm were cut, dried and counterstained with Mayer's haematoxylin. Tumour epithelial cells were microdissected on to caps (CellPix II, Arcturus), DNA was extracted as above and PCR was repeated on these samples. For each case, two separate samples composed purely of tumour cells were taken, one containing approximately 100 cells and one with approximately 300 cells. Tissue was also taken from adjacent normal or dysplastic epithelium where applicable.

Results

None of the 24 controls tested positive for HPV DNA by real‐time PCR whereas 10 of 101 (10%) cases did. Two cases from the laryngeal group yielded weak HPV DNA PCR‐positive results, which could not be repeated. These two cases did not test HPV positive after application of the PGMY primer set, and therefore could not be genotyped. Eight cases derived from Waldeyer's ring contained HPV DNA by real‐time PCR. All eight were strong, repeatable positives and genotyping was possible in every case.

The mean age in the 10 positive cases was 50.6 years (95% CI 44.11 to 57.09). The mean age in the 91 HPV‐negative cases (excluding controls) was 63.3 years (95% CI 61.17 to 65.5). This age difference is significant (unpaired Student's t test, t = −3.69, p<0.001).

HPV genotyping showed that 7 of 8 strong‐positive cases contained HPV type 16. The eighth strong‐positive case contained HPV type 11. No mixed infections were detected (all 8 positives tested positive for β globin; table 11).

Table thumbnail
Table 1 Analysis of human papilloma virus‐positive tumour biopsy specimens

HPV DNA was not detected in microdissected tissue from the two weakly positive carcinomas from the larynx. Of the eight cases from Waldeyer's ring, five tested positive for HPV DNA when microdissected (table 22).). HPV DNA was detected in one of three microdissected samples of adjacent dysplastic epithelium. The adjacent epithelium in case 7 had a non‐dysplastic, warty appearance and this epithelium and the background normal epithelium contained HPV DNA.

Table thumbnail
Table 2 Human papilloma virus PCR results in microdissected samples from positive cases

Discussion

A previous study of HPV in head and neck carcinoma in a UK population concentrated on oral lesions, with HPV found in up to 46% of normal and tumour tissues.10 We have investigated the presence of HPV in tonsillar and laryngeal carcinomas, and our findings replicate the results of similar studies performed in other countries, confirming that any future HPV‐directed diagnostic and treatment strategies for HNSCC are relevant to this UK‐based population. We have confirmed that HPV seems to have a predilection for involvement in carcinomas arising in Waldeyer's ring, suggesting that detection of HPV in this subset of tumours may be of clinical relevance not least because HPV‐positive carcinomas seem to have a better prognosis, possibly because of a greater sensitivity to radiation treatment than conventional HNSCC.11,12 Paradoxically, a recent study found that HPV‐positive HNSCCs were associated with earlier nodal metastasis and suggested that screening high‐risk populations for HPV may be valuable in preventing premalignant lesions progressing to carcinoma.13

Microdissection confirmed that HPV DNA was present in carcinoma cells. The detection of HPV in some of the small microdissected samples has shown the sensitivity of these techniques in formalin‐fixed paraffin wax‐embedded material, which is of relevance, as biopsy specimens submitted from head and neck sites may be limited in size.

The presence of HPV DNA in abnormal epithelium adjacent to the invasive carcinoma in two cases and normal epithelium in one case suggests that the detection of HPV in the epithelium of Waldeyer's ring could indicate an increased risk of neoplastic transformation with possible future diagnostic utility, as is being developed for the cervix. Previous studies that investigated the presence of HPV DNA in normal head and neck epithelium found wide‐ranging results,14 and therefore further work in this area is necessary before HPV testing can be used clinically to identify patients who are potentially at risk of head and neck malignancy.

Acknowledgements

This work was supported by a grant from the Lothian University Hospitals NHS Trust Cancer Services Committee. We thank Dominic Rannie for his guidance in the use of the laser capture microscope.

Abbreviations

HNSCC - head and neck squamous cell carcinoma

HPV - human papillomavirus virus

PCR - polymerase chain reaction

Footnotes

Competing interests: None.

Ethical approval: This study received ethical approval from the Lothian Research Ethics Committee, Edinburgh, UK.

References

1. Haraf D J, Nodzenski E, Brachman D. et al Human papilloma virus and p53 in head and neck cancer: clinical correlates and survival. Clin Cancer Res 1996. 2755–762.762 [PubMed]
2. Paz I B, Cook N, Odom‐Maryon T. et al Human papillomavirus in head and neck cancer. An association of HPV 16 with squamous cell carcinoma of Waldeyer's tonsillar ring. Cancer 1997. 79595–604.604 [PubMed]
3. Klussmann J P, Weissenborn S J, Wieland U. et al Human papillomavirus‐positive tonsillar carcinomas: a different tumor entity? Med Microbiol Immunol (Berl) 2003. 192129–132.132 [PubMed]
4. El‐Mofty S K, Lu D W. Prevalence of human papillomavirus type 16 DNA in squamous cell carcinoma of the palatine tonsil, and not the oral cavity, in young patients: a distinct clinicopathologic and molecular disease entity. Am J Surg Pathol 2003. 271463–1470.1470 [PubMed]
5. Devaraj K, Gillison M L, Wu T C. Development of HPV vaccines for HPV‐associated head and neck squamous cell carcinoma. Crit Rev Oral Biol Med 2003. 14345–362.362 [PubMed]
6. Cubie H A, Seagar A L, McGoogan E. et al Rapid real time PCR to distinguish between high risk human papillomavirus types 16 and 18. Mol Pathol 2001. 5424–29.29 [PMC free article] [PubMed]
7. de Roda Husman A M, Walboomers J M, van den Brule A J. et al The use of general primers GP5 and GP6 elongated at their 3 ends with adjacent highly conserved sequences improves human papillomavirus detection by PCR. J Gen Virol 1995. 761057–1062.1062 [PubMed]
8. Gravitt P E, Peyton C L, Alessi T Q. et al Improved amplification of genital human papillomaviruses. J Clin Microbiol 2000. 38357–361.361 [PMC free article] [PubMed]
9. Gravitt P E, Peyton C L, Apple R J. et al Genotyping of 27 human papillomavirus types by using L1 consensus PCR products by a single‐hybridization, reverse line blot detection method. J Clin Microbiol 1998. 363020–3027.3027 [PMC free article] [PubMed]
10. Maitland N J, Cox M F, Lynas C. et al Detection of human papillomavirus DNA in biopsies of human oral tissue. Br J Cancer 1987. 56245–250.250 [PMC free article] [PubMed]
11. Ritchie J M, Smith E M, Summersgill K F. et al Human papillomavirus infection as a prognostic factor in carcinomas of the oral cavity and oropharynx. Int J Cancer 2003. 104336–344.344 [PubMed]
12. Lindel K, Beer K T, Laissue J. et al Human papillomavirus positive squamous cell carcinoma of the oropharynx: a radiosensitive subgroup of head and neck carcinoma. Cancer 2001. 92805–813.813 [PubMed]
13. Hoffmann M, Gorogh T, Gottschlich S. et al Human papillomaviruses in head and neck cancer: 8 year‐survival‐analysis of 73 patients. Cancer Lett 2005. 218199–206.206 [PubMed]
14. McKaig R G, Baric R S, Olshan A F. Human papillomavirus and head and neck cancer: epidemiology and molecular biology. Head Neck 1998. 20250–265.265 [PubMed]

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