• Carlos Eduardo Xavier dos Santos Ribeiro da Silva
• Ismael Dale Cotrim Guerreiro da Silva
• Artur Cerri
• Luc Louis Maurice Weckx Oral Surgery
• Oral Medicine
• Oral Pathology
• Oral Radiology and Endodontics
• October 2007 (Vol. 104, Issue 4, Pages 497-500)

ABSTRACT

Oncogenic human papillomaviruses (HPVs) are important agents in the genesis of gynecological cancer, and have also been implied in the genesis of oral cancer. With the purpose of evaluating the relation between HPV and squamous cell carcinoma (SCC) of the tongue a case-control study was performed. 50 Caucasian male patients who were smokers and had the histological diagnosis of SCC of the tongue were selected. The control group was comprised of 10 matched patients with no clinical evidence of tongue lesions. Polymerase chain reaction (PCR) was used to detect the presence of HPV genome in fresh-frozen tissue specimens from SCC of the tongue margin. Thirty seven patients (74%) had a positive PCR for oncogenic papillomavirus, and only one specimen (10%) of the control group was positive for non-oncogenic papillomavirus. Based on the statistical analysis of this study there was a 25.6 higher risk for SCC of the tongue to harbor oncogenic HPV than the healthy control tongur tissue.

INTRODUCTION

Human papillomavirus (HPV) is a virus of the Papillomavirus genus, of the Papovaviridae family, with more than 120 subtypes identified up to the moment, and is composed of a genome of 8000 base pairs of double-stranded DNA forming a complex similar to a chromosome encased by a 55nm non-enveloped outer capsid protein.1 The cleavage site of viral DNA circular molecules is specific, that is, it is always cleaved at the same site between E1 and E2 genes. E2 is responsible for repressing transcription of the E6 and E7 viral genes. Once E2 is inactivated by cleavage of the viral molecule, a dysregulated expression of the E6 and E7 genes occurs.1

The oncogenic potential of the HPV is related to gene products that interact and inactivate cell proteins derived from suppressor genes of p53 and p105-RB and promote degradation of these genes, thus blocking their function. Oncogenicity, among other conditions, will depend directly on the degree of affinity between proteins derived from tumor suppressor genes and viral proteins derived from the E6 and E7 genes. 1 Thus, “high risk� E6 and E7 gene products have a high affinity for p53 and p105-RB derived proteins, whereas “low risk� viral gene derived products have a low affinity for these proteins. The result of viral penetration is the immortalization of cells in which HPV had integrated. These cells morphologically exhibit abnormal mitosis, nuclear pleomorphism, aneuploid DNA content consistent with abnormal chromosome number, and chromosome architectural changes.1 However, these cells only start to generate tumors when the E6 and E7 transforming genes are exposed to activated cell oncogenes1. However, HPV may not operate alone in oncogenesis. In theory, other factors such as the host’s immune status, nutritional deficiency, and cigarette and alcohol consumption operate together favoring and potentializing tumor onset. The association of these factors with oncogenic HPV subtypes is extremely important in the genesis of malignant tumors, since cigarette and alcohol operate as inducing factors and papillomavirus operates in the tumor progression phase.

In fact, recent evidence suggests that HPV can be present in up to 100% of patients with cervical carcinoma.2 The different types of HPV are divided into two groups depending on their oncogenic potential. HPVs considered non-oncogenic are the 6, 11, 42 and 54 subtypes, usually present in verrucous lesions, papillomas and condylomas.3 The oncogenic subtypes are the 16, 18, 31, 33, 35, 39, 45, 51, 55, 56, 58, 66, and 68, which are frequently found in patients with malignant neoplasms.3,4 The mere presence of oncogenic HPV may increase the relative potential for the development of cervical intraepithelial neoplasm by up to 116 times.5 The Word Health Organization considers HPV a carcinogen. Oral cavity cancer is a major cause of mortality and morbidity in Brazil. According to INCA (Brazilian National Cancer Institute)6 data, the estimated incidence of new cases for 2005 ranks it as the eighth most frequent type of cancer in males and ninth in females, with 9985 and 3895 cases, respectively. The most prevalent type of cancer of the oral cavity is the squamous cell carcinoma (SCC) accounting for approximately 95% of all malignant neoplasms of this site. The principal risk factors for oral cancer are smoking, excessive alcohol consumption, and exposure to sun radiation (for neoplasm of the lower lip).

Some authors have associated the presence of HPV, particularly of type 16 as factors contributing to the onset of squamous cell carcinoma (SCC) of the ororpharynx, but its role in oral cavity cancer is controversal.7,8 and 4 The prevalence of HPV in oral cavity cancer has been variable in the literature, mainly due to variations in site and mixture with oropharynx sample size, population studied and sensitivity of the techniques used. The previous results were very debatable and we believed that the methods used in previous research were inadequate. Such studies have used in situ hybridization and the hybrid capture as HPV detection methods, and such methods are substantially less sensitive than PCR. Besides, while the fresh-frozen tissue has a higher preservation rate of viral DNA, many studies have used material from paraffin blocks. A study conducted in 1996 demonstrated that the presence of HPV is more frequent when the sample is tested by PCR (37,1%) than when tested by in situ hybridization (16,9%) and that it is also more frequent when frozen oral tissue is tested (51,6%) than when paraffin-embedded tissue is (21,7%).9Â A meta-analysis of 4680 samples of oral SCC has come to the conclusion that the presence of HPV is more frequent in carcinomatous epithelium and is an independent significant risk factor for oral SCC.10 The objective of the present study is to evaluate the presence of human papillomavirus (HPV) genome both oncogenic and non-oncogenic types in fresh tissue specimens from oral tongue SCC, using polymerase chain reaction (PCR), and to compare these results with those obtained in tissue specimens of a similar site removed a control group of cancer-free individuals.

METHODS

Sixty fresh tissue specimens from the oral tongue were used in this study. Fifty were from patients with squamous cell carcinoma (SCC) of the oral tongue and 10 were from volunteers ( from the hospital staff ) of the control group who had no clinical evidence of disease. and no one had history of cancer The patients were selected at the Outpatient Service of Oral Medicine, of the Federal University of Sao Paulo – UNIFESP, and at the Oupatient Service of the Discipline of Oral Medicine of the Santo Amaro University – UNISA. The material was collected only after approval of the Research Ethics Committees of both UNIFESP and UNISA. All of those patients readied and signed the informed consent.

All subjects participating in the study were males, older than 40 years, Caucasian and smokers. These criteria were used because they are statistically related to the group with a higher incidence of oral cavity cancer according to the Brazilian National Cancer Institute.6 We performed anesthesia followed by incisional biopsy of the lesion, from which a small fragment of tissue was removed using a number-5 biopsy punch (diameter of approximately 5.0mm). For patients with no clinical signs of disease (control group), a small amount of anesthetic agent was infiltrated in the lateral oral tongue, and the tissue was removed with a number-2 biopsy punch (diameter of 2.00 mm).

The tissue removed with a diagnostic hypothesis of SCC was divided into two fragments, one of which was immersed in 10% formalin for pathological examination to confirm the diagnosis, and the other was immersed in an Eppendorf tube with 0.9% saline solution and frozen at minus 20ºC in the Laboratory of Molecular Gynecology of UNIFESP. A similar method was used for the control group. For the SCC group, all patients had squamous cell carcinoma confirmed by histopathological examination.

The “Phenol-Chloroformâ€? protocol for DNA extraction was used for the 60 fresh frozen specimens collected (50 in the SCC group and 10 in the control group). For polymerase chain reaction (PCR), a commercial kit for the detection of HPV (BIOPAP kIT) was used. BIOPAP KIT is manufacturated by Biotools B & M Labs S.A. – Madrid, Spain. According to the manufacturer this kit is able to detect the presence of HPV and separate it into two groups named oncogenic – 16, 18, 31, 33, 35, 52, 58 and 67 and non-oncogenic – 6, 11, 13, 16, 18, 30, 31, 32, 33, 34, 35, 39, 40, 42, 43, 44, 51, 52, 53, 54, 55, 56, 57, 58, 59, 61, 62, 64, 66, 67, 68 and 69   (manufacture’s nomenclature) according to its oncogenic potential. It needs only 10 copies of the HPV DNA to be positive. For each assay a negative control (DNA replaced by water) and another molecular weight marker were amplified in addition to the specimens and the positive control included in the commercial kit was performed to certificate the method. The visualization of the bands under ultraviolet transillumination enabled determining the presence or absence of HPV DNA in the specimens. In the cases where the reaction was positive thanks to the difference in the number of base pairs between the “oncogenicâ€? (250bp) and the “non-oncogenicâ€? (450 bp) groups, the classification of the specimens in one of these groups was also possible.

The Fisher’s exact test was used to evaluate whether the presence of HPV was associated with oral cancer. To quantify this association, the odds ratio was estimated whenever possible, that is, the likelihood of a patient with HPV to have oral cavity cancer when compared to a patient without HPV using a univariate logistic regression model. The response variable in this model was the presence of cancer (yes or no), and the explanatory variable was the presence of HPV (yes or no).

RESULTS

Of the 50 specimens of squamous cell carcinoma of the lateral oral tongue analyzed by polymerase chain reaction (PCR) to detect human papillomavirus DNA, 37 (74%) were positive for the “oncogenic� subtypes. None of the specimens (0%) was exclusively positive for “non-oncogenic� subtypes. Of the 10 specimens of normal tissue from the control group analyzed with this method, only one (10%) was positive for “non-oncogenic� subtypes and no specimens (0%) were positive for “oncogenic� subtypes.

The prevalence of HPV was not similar in the two groups (p≤0.01). Logistic regression model analysis demonstrate that patients with SCC of the tongue had 25.6 higher odds (95%CI = 3.0 – 222.2) of having oncogenic HPV than the controls. Since none of the patients without oral cancer had oncogenic HPV, estimates using the logistic regression model for odds ratio could not be obtained.Â

DISCUSSION

An interesting study has demonstrated the presence of HPV 16 in 69.2% of oral SCC cases and of HPV 18 in 28% of them by utilizing a combination of PCR with in situ hybridization and has shown also that the same samples tested negative when tested with isolated hybridization11. In our study we have used two groups with different numbers of individuals, being 50 with tongue SCC and 10 without the disease, following the orientation of the Research Ethics Committee – regarding biopsy in patients with no evidence of disease. Despite the different sizes of the material collected in the biopsies (5 mm for SCC cases and 2 mm for the control group), the material used in the PCR had practically the same size, for the fragments of the SCC cases were cut in half to allow the histopathological analysis. We observed that out of a total of 50 specimens of squamous cell carcinoma removed from the lateral oral tongue, 74% were positive for oncogenic subtypes of HPV. These results are similar to those obtained by other authors: 78%12;72.5%;13 72.5%14; 91%15, and 74%16. These results suggest that oncogenic HPV should be considered as a risk factor for oral tongue cancer.

On the other hand, our results are not in agreement with those obtained by several authors such as Chang et al. (1990) who found HPV in 27.5%17 of the oral cavity SCC specimens analyzed; and other authors in 8.4%18; 31%19; 12.5%20 and 42%21. The discrepancy of results is attributed to the fact that in most cases in which the prevalence of HPV was low, a viral DNA amplification from paraffin blocks was performed, whereas in the studies where the prevalence of HPV was higher, like in ours, the DNA was amplified from fresh tissue (frozen immediately after biopsy). The material is usually fixed in 10% formalin prior to being embedded in a paraffin block. Formalin fixation, paraffin embedding, and subsequent deparaffinization can lead to degradation of viral DNA making its detection impossible or difficult9. Secondly, the high-sensitivity and specificity BIOPAP kit (Spain) was used for this study. This kit has the property to detect the presence of HPV genome and to discriminate its potential into the oncogenic and non-oncogenic groups.

Of the 10 specimens of the control group which presented a clinically normal mucosa, only one (10%) was positive for the non-oncogenic subtype, and none of the specimens was positive for oncogenic subtypes. In this sense, our study corroborates many other studies conducted by this authors with 0%14; 11.1%22; and 0%20. Chang et al17. (1990) analyzed 40 specimens of normal tissue and found no evidence of HPV. Based on our findings and on the analysis of other authors’ studies we believe that the tongue is the not the only place in the mouth where oncogenic HPV can be found and we believe that other places in the mouth may have the SCC risk increased by the presence of HPV.

CONCLUSIONS

The study conducted with fresh-frozen specimens of oral tongue squamous cell carcinoma using PCR allowed us to conclude that oncogenic human papillomavirus is present in 74% of cases, whereas the control group had a prevalence of none (only 10% non-oncogenic). These rates suggest that oral cavity cancer HPV positive rates may be higher than previously suspected. More studies are needed to know if the simple presence of HPV in oral cavity can increase the risk of oral cancer.

KEYWORDS: Papillomavirus, Mouth Neoplasms, Head and Neck Neoplasms, Oncogenic viruses, Polymerase Chain Reaction

REFERENCES

1. Syrjanen K, Syrjanen S, Lamberg M, Pyrhonen S, Nuutinen J. Morphological and immunohistochemical evidence suggesting human papillomavirus (HPV) involvement in oral squamous cell carcinogenesis. Int J Oral Surg 1983;dec;12(6):418-24.

2. zur Hausen H. Papillomavirus causing cancer: evasion from host-cell control in early events in carcinogenesis. J Natl Cancer Inst. 2000; 92(9), 690-8.

3. Villa LL. Human papillomavirus and cervical cancer. Advanc Canc Res. V.71, p.321-341, 1997

4. Watts SL; Brewer EE; Fry TL. Human papillomavirus types in squamous cell carcinomas of the head and neck. Oral Surg Oral Med Oral Pathol; 71(6):701-7, 1991 Jun.

5. Rozendal AL, Walboomers JMM, van der Linden JC, Voorhost FJ, Kenemans P, Helmerhost TJM, Ballegooijen M. PCR based high-risk HPV test in cervical cancer screening gives objective risk assessment of women with cytomorphologically normal cervical smears. Int J Cancer; v.68, p.766-9, 1996.

6. Brazilian National Cancer Institute www.inca.gov.br/cancer. Access in 15/04/2006.

7. Gillison, ML et al. Evidence for a causal association between human papillomavirus and a subset of head and neck cancers. Journal of the National Cancer Institute 2000;92(9):709-20.

8. Mineta H, Ogino T, Amano HM, Ohkawa Y, Araki K, Takebayashi S, Miura K. Human papilloma virus (HPV) type 16 and 18 detected in head and neck squamous cell carcinoma. Anticancer Res 1998;nov-dec;18(6B):4765-8.

9. Miller CS, White DK. Human papillomavirus expression in oral mucosa, premalignant conditions, and squamous cell carcinoma: a retrospective review of the literature. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 1997 Feb;83(2):187-8

10. Miller CS; Johnstone BM. Human papillomavirus as a risk for oral squamous cell carcinoma: a meta-analysis, 1982-1997. Oral Surg Oral Med Oral Pathol Oral Radiol Endod; 91(6):622-35, 2001 Jun.

11. Miller CS; Zeuss MS; White DK. Detection of HPV DNA in oral carcinoma using polymerase chain reaction toguether with in situ hybridization. Oral Surg Oral Med Oral Pathol; 77(5):480-6, 1994 May

12. Woods KV, Shillitoe EJ, Spitz MR, Schantz SP, Adler-Storthz K. Analysis of human papillomavirus DNA in oral squamous cell carcinomas. J Oral Pathol Med 1993;mar;22(3):101-8.

13. Nielsen H, Norrild B, Vedofte P. Human papillomavirus in oral premalignant lesions. Eur j Cancer B Oral Oncol 1996;(32):264-70.

14. Cao J, Zhang ZY, Zhang YX. Human papillomavirus infection and p 53 alteration in oral squamous cell carcinoma. Chin Dent Res 2000;nov;3(3):44-9.

15. Bouda M, Gorgoulis VG, Kastrinakis NG, Giannoudis A, Tsoli E, Danassi-Afentaki D, et al. “High risk” HPV types are frequently detected in potentially malignant and malignant oral lesions, but not in normal oral mucosa. Mod Pathol 2000;jun;13(6):644-53.

16. Balaram P, Nalinakumari KR, Abraham E, Balan A, Hareendran NK, Bernard HU, Chan SY. Human papillomaviruses in 91 oral cancers from Indian betel quid chewers-high prevalence and multiplicity of infections. Int J Cancer 1995;may; 16; 61(4):450-4.

17. Chang F, Syrjanen S, Nuutinen J, Karja J, Syrjanen K. Detection of human papillomavirus (HPV) DNA in oral squamous cell carcinomas by in situ hybridization and polymerase chain reaction. Arch Dermatol Res 1990;282(8):493-7.

18. Kansky AA, Poljak M, Seme K, Kocjan BJ, Gale N, Luzar B, Golouh R. Human papillomavirus DNA in oral squamous cell carcinomas and normal oral mucosa. Acta Virol 2003;47(1):11-6.

19. Koppikar P, deVilliers EM, Mulherkar R. Identification of human papillomaviruses in tumors of the oral cavity in an Indian community. Int J Cancer 2005;mar;113(6):946-50.

20. Sand L, Jalouli J, Larsson PA, Hirsch JM. Human papilloma viruses in oral lesions. Anticancer Res 2000;mar-apr;20(2B):1183-8.

21. Ibieta BR, Lizano M, Fras-Mendivil M, Barrera JL, Carrillo A, Ma Ruz-Godoy L, Mohar A. Human papilloma virus in oral squamous cell carcinoma in a Mexican population. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2005;mar;99(3):311-5.Â

22. Summersgill KF, Smith EM, Kirchner HL, Haugen TH, Turek LP. p53 polymorphism, human papillomavirus infection in the oral cavity and oral cancer. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2000;90(3):334-9.