In situ hybridization for human papillomavirus DNA in squamous cell carcinomas of the larynx and inverted papillomas

Introduction
Head and neck cancers are among the most common cancers worldwide and are responsible for 350 000 deaths each year. Nearly 90% of these cancers are squamous cell carcinomas [1, 2]. Head and neck malignancies are characterized by a multiphasic and multifactorial aetiopathogenesis. Smoking and alcohol consumption are the most common risk factors for oral, oropharyngeal, and laryngeal carcinomas. There is increasing molecular and epidemiological evidence that human papillomaviruses (HPVs) may act as a cocarcinogen, along with tobacco, in the causation of these cancers [3, 4].
The role of HPV in the aetiology of anogenital cancers has been firmly established, and infection with this virus has also been shown to have prognostic significance [5, 6]. The similarity of the morphological features of genital and oral HPV-associated lesions was one of the early findings that raised the possibility that HPV might be involved in oral and laryngeal squamous cell carcinomas [7, 8]. Between 15% and 35% of head and neck squamous cell cancers could be associated with high risk HPVs, in particular HPV 16 [9, 10].
Infections with HPV types that have low oncogenic risk, such as HPV 6 and 11, are associated with benign lesions of the anogenital areas known as condylomata acuminata (genital warts), low-grade squamous intraepithelial lesions of the cervix as well as head and neck papillomas [11, 12]. Human papillomavirus 6 and 11 are the most frequently identified HPV subtypes in oral and sinonasal inverted papillomas [13-16]. Inverted papillomas are particularly noteworthy because in contrast to exophytic papillomas, they are associated with malignancy and have a tendency to recur.
Infections with HPVs are divided basically into three different infection types: those producing specific clinically visible lesions, those remaining subclinical, and those being latent. Histopathological examinations are usually sufficient to reveal characteristic signs of HPV infection including: binucleation, multinucleation, koilocytosis, spindle koilocytes, abnormal mitosis, increased mitotic activity, dyskeratosis and parakeratosis. The most popular viral cytopathic effect of HPV infection is koilocytosis, which is considered a major criterion in HPV infection from the histopathological point of view [17]. Although it is known that molecular methods are the best current method to detect HPVs, the diagnosis is usually established by investigating morphological signs of HPV infection with haematoxylin and eosin stained slides.
The aim of our study was to determine whether an association exists between histopathological signs of HPV infection and presence of HPV DNA in cases of squamous cell carcinomas and inverted papillomas with and without morphological signs of HPV infection.

Material and methods
Patients
Twenty-six formalin-fixed, paraffin-embedded tissue specimens of squamous cell carcinoma of the larynx and 17 of sinonasal inverted papillomas were retrieved from archival material (Chair of Pathomorphology, Medical University of Lodz). The age range for sinonasal inverted papillomas was from 29 to 79 years (mean 53.59) and for squamous cell carcinomas of the larynx was from 47 to 82 years (mean 62.77). Paraffin tissue sections were stained with haematoxylin and eosin and the histological diagnoses were established according to WHO classification [18]. Ten out of 26 cases of squamous cell carcinoma of the larynx and 11 out of 17 sinonasal inverted papillomas showed histological signs of HPV infections (koilocytosis, binucleated squamous cells, increased mitotic activity and abnormal mitoses).

In situ hybridization
Seventeen sinonasal inverted papillomas and 26 squamous cell carcinomas of the larynx were analysed using commercially available HPV DNA probes (Dako, Carpinteria, California, USA). Initially, a wide spectrum biotinylated probe for common HPV subtypes was used, according to the manufacturer’s suggested protocol. The wide spectrum probe targets the genomic DNA of HPV types 6, 11, 16, 18, 31, 33, 35, 39, 45, 51 and 52. Further subtyping was carried out in the same way, using specific probes for HPV low risk (HPV 6 and 11) and HPV high risk (HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68). Afterwards a catalysed signal amplification system prepared according to the instructions of the manufacturer was used (GenPoint, CSA System for in situ hybridization; Dako).

Statistical methods
The differences between groups were tested using non-parametric 2 test. Results were considered statistically significant if p < 0.05.
Results
Of the 11 cases of sinonasal inverted papillomas with morphological signs of HPV infection, 1 was positive for HPV DNA using the wide spectrum probe and for HPV DNA subtypes 6 and 11 (Fig. 3). In addition, 1 case was positive for the HPV high risk probe and 1 for the wide spectrum HPV probe only. None of the 6 cases of sinonasal inverted papillomas without morphological signs of HPV infection was positive for any HPV DNA probes.
One of the 10 cases of squamous cell carcinoma with morphological signs of HPV infection was positive for both HPV DNA probes: the wide spectrum probe and the HPV high risk probe. In addition, positive reactions for HPV DNA using the high risk probe were noted in 3 examined cases of squamous cell carcinomas of the larynx where the morphological signs of HPV infection were present (Fig. 1). Four of the 16 cases of squamous cell carcinoma without morphological signs of HPV infection were positive for both HPV DNA probes – the wide spectrum probe and the high risk probe (Fig. 2) – and 1 case was positive for the high risk probe only.
No significant differences in the frequency of HPV infection were noted between the two examined groups (with and without morphological signs of HPV infection) of sinonasal inverted papillomas (p = 0.15) or between the two groups of squamous cell carcinomas of the larynx (p = 0.64).
The results of in situ hybridization are summarized in Table I.

Discussion
Human papillomavirus infection of human tissues is reflected in a spectrum of histological changes. According to many authors, koilocytosis is a patho­gnomonic sign of HPV infection serving as a foun­dation for molecular biology studies. Although molecular methods are available for detecting HPV infections, the presence of morphological features of HPV infection in tissues is still fundamental for histopathological diagnosis. It is a routinely used method particularly in centres in which sophisticated molecular methods are unavailable. Inverted papillomas have been studied extensively for their association with HPV 6 and HPV 11 [13-16]. Positive reaction for a low risk HPV probe, found in our study in 1 case with morphological signs of HPV infections, may suggest the possible involvement of HPV 6 and HPV 11 in aetiology of sinonasal inverted papillomas. It is well known that inverted papillomas are lesions that have a tendency for transformation into squamous cell carcinoma. Many authors suggest that high risk HPV, particularly HPV 16, may be involved in malignant transformation of inverted papillomas [19-21]. The positive reaction for the high risk HPV probe observed in our study may support the hypothesis that HPV infection may play a role in the progression of inverted papillomas to carcinomas. Lack of the expression of HPV DNA in the remaining cases of inverted papillomas with and without morphological features of HPV infection could be caused by the presence of HPV subtypes other than those investigated in our study. Guan [22] observed that HPV 57 may be related to the pathogenesis of nasal inverted papillomas. It is also possible that the negative results in both tested groups of inverted papillomas could reflect the aetiology of these lesions a diffe­rent than viral. The role of HPV in the pathogenesis of inverted papillomas is still questionable and some analysis suggests that incidental expression of HPV may occur as well. For instance, Syrjänen [2] demonstrated HPV in approximately 11% of cases of normal oral mucosa.
Our results suggest involvement of HPV in pathogenesis of squamous cell carcinoma of the larynx. The 40% positivity rate of high risk HPV subtypes in tissue samples from patients with squamous cell carcinoma with morphological signs of HPV infection observed in our study is similar to that seen in other studies [8, 13, 23, 24]. It is worth noting that even though morphological signs of HPV infections were present in all tested samples, HPV DNA was detected only in some of the examined samples. The literature data suggest that HPV 16 and HPV 18 are the main HPV subtypes aetiologically involved in the development of squamous cell carcinomas of the larynx [2, 13, 25]. In contrast to other findings, we observed 3 positive results with the high risk HPV probe only. It is possible that subtypes of HPV 56, 58, 59, 68, which are not present in the wide spectrum probe, may play a role in pathogenesis of squamous cell carcinoma in these cases. It is well known that the aetiology of squamous cell carcinomas of the larynx is complex, also demographic and individual variables exists. In our opinion, this is one of the reasons for the highly variable detection of HPV DNA.
In the group of cancers without morphological signs of HPV infections, we obtained unexpected findings. Four of the 16 cases of squamous cell carcinoma were positive for both the wide spectrum HPV probe and the high risk HPV probe. Additionally, 1 case was positive for the high risk probe only. Our results support the presumption of HPV involvement in the pathogenesis of squamous cell carcinomas of the larynx in cases without morphological signs of HPV infections as well. To our knowledge, this is the first study evaluating the frequency of HPV DNA in cancers without signs of HPV infection. It must be taken into consideration that tissues for HPV studies are usually qualified with special emphasis on the presence of morphological signs suggesting HPV infection. Absence of morphological signs of HPV infection in cases with positive HPV DNA expression provides interesting insights into the histopathological value of diagnosis based on routinely stained samples. On the other hand, our results may point, on the one hand, to underestimation of HPV infection in cases without morphological signs of HPV infection but positive for HPV DNA, and to overestimation in cases with morphological signs of HPV infection but negative for HPV DNA. In our opinion the histopathological analyses of haematoxylin and eosin stained tissues are not sufficient for accurate evaluation of HPV infection and should be a screening method of HPV infection only.
In summary, our study confirms the role of HPVs in the pathogenesis of sinonasal inverted papillomas and laryngeal squamous cell carcinoma. However, we also demonstrated the existence of relevant discrepancies between the presence of HPV DNA and morphological signs of HPV infection, because only in some of the examined cases morphological signs of HPV infection were associated with the presence of HPV DNA.

Acknowledgments
This study was supported by the Medical University of Lodz, grant 502-16-822.

References  
1. Kim ES, Kies M, Herbst RS. Novel therapeutics for head and neck cancer. Curr Opin Oncol 2002; 14: 334-342.  
2. Syrjänen S. Human papillomavirus (HPV) in head and neck cancer. J Clin Virol 2005; 32: 59-66.  
3. Mork J, Lie AK, Glattre E, et al. Human papillomavirus infection as a risk factor for squamous-cell carcinoma of the head and neck. N Engl J Med 2001; 344: 1125-1131.  
4. Chocolatewala NM, Chaturvedi P. Role of human papilloma virus in the oral carcinogenesis: an Indian perspective. J Cancer Res Ther 2009; 5: 71-77.  
5. Burk RD, Chen Z, Van Doorslaer K. Human papillomaviruses: genetic basis of carcinogenicity. Public Health Genomics 2009; 12: 281-290.  
6. Lehoux M, D'Abramo CM, Archambault J. Molecular mechanisms of human papillomavirus-induced carcino­genesis. Public Health Genomics 2009; 12: 268-280.  
7. IARC 1995. Monographs on the evaluation of carcinogenic risks to human. Papillomaviruses (www.inchem.org/ documents/iarc/vol64/hpv.html)  
8. Major T, Szarka K, Sziklai I, et al. The characteristic of human papillomavirus DNA in head and neck cancer and papillomas. J Clin Pathol 2005; 58: 51-55.  
9. Slebos RJ, Yi Y, Ely K, et al. Gene expression differences associated with human papillomavirus status in head and neck squamous cell carcinoma. Clin Cancer Res 2006; 12 (3 Pt 1): 701-709.
10. Neufcoeur PE, Arafa M, Delvenne P, et al. Involvement of human papillomavirus in upper aero-digestive tracts cancers. Bull Cancer 2009; 96: 941-950 (abstract).
11. Bosch FX, Lorincz A, Mun~oz N, et al. The casual relation between human papilomavirus and cervical cancer. J Clin Pathol 2002; 55: 244-265.
12. Hsueh PR. Human papillomavirus, genital warts, and vaccines. J Microbiol Immunol Infect 2009; 42: 101-106.
13. Brandwein M, Steinberg B, Thung S, et al. Human papillomavirus 6/11 and 16/18 in schneiderian inverted papillomas, In situ hybridization with human papillomaviruses RNA probes. Cancer 1989; 63: 1708-1713.
14. Zhou Y, Hu M, Li Z. Human papillomavirus (HPV) and DNA test in inverted papillomas of the nasal cavities and paranasal sinuses. Zhonghua Er Bi Yan Hou Ke Za Zhi 1997; 32: 345-347 (abstract).
15. Kashima HK, Kessis T, Hruban RH, et al. Human papillomavirus in sinonasal papillomas and squamous cell carcinoma. Laryngoscope 1992; 102: 973-976.
16. Batsakis JG, Suares P. Schneiderian papillomas and carcinomas: a review. Adv Anat Pathol 2001; 8: 53-64.
17. Krawczyk E, Suprynowicz FA, Liu X, et al. Koilocytosis: a cooperative interaction between the human papillomavirus E5 and E6 oncoproteins. Am J Pathol 2008; 173: 682-688.
18. Barnes L, Everson JW, Reichart P, et al. World Health Organization Classification of Tumours. Pathology and Genetics Head and Neck Tumours. IARC Press, Lyon 2005; 26-32, 118-121.
19. Lu SS, Zou R, Xu JW. Relationship between prognosis of nasal inverted papilloma and human papillomavirus types. Zhonghua Er Bi Yan Hou Tou Jing Wai Ke Za Zhi 2005; 40: 195-198 (abstract).
20. McKay SP, Grégoire L, Lonardo F, et al. Human papillomavirus (HPV) transcripts in malignant inverted papilloma are from integrated HPV DNA. Laryngoscope 2005; 115: 1428-1431.
21. Kim JY, Yoon JK, Citardi MJ, et al. The prevalence of human papilloma virus infection in sinonasal inverted papilloma specimens classified by histological grade. Am J Rhinol 2007; 21: 664-669.
22. Guan Y, Zang C, Zhang C. Study on correlation between HPV57 infection and the etiology of IP. Lin. Chuang Er Bi Yan Hou Ke Za Zhi 2006; 20: 922-924, 927 (abstract).
23. Hartley C, Hamilton J, Birzgalis AR, et al. Recurrent respiratory papillomatosis – the Manchester experience, 1974-1992. J Laryngol Otol 1994; 108: 226-229.
24. Syrjänen S. Human Papillomaviruses in Head and Neck Carcinomas. N Engl J Med 2007; 357: 313.
25. Al Moustafa AE, Kassab A, Darnel A, et al. High-risk HPV/ErbB-2 interaction on E-cadherin/catenin regulation in human carcinogenesis. Curr Pharm Des 2008; 14: 2159-2172.


Address for correspondence
Olga Stasikowska-Kanicka MSc, PhD
Department of Nephropathology
Medical University of Lodz
ul. Czechosłowacka 8/10
92-216 Łódź
phone/fax +48 42 679 01 91
e-mail:olgast@op.pl