INTRODUCTION

Oncogenic human papillomavirus (HPV) has been well associated with anogenital and cervical cancer, however, the correlation between HPV and oral squamous cell carcinoma has not been properly defined yet. The possible correlation of HPV in the etiology of cancer damage and oral cancer was first estimated in 1983, when authors described the cytopathic abnormalities of HPV (coilocytes) in oral cancer, identical to that in pre-malignant and cervical cancer cases 1. Throughout the years, many studies investigating the role of HPV in carcinoma and other potentially malignant oral lesions have been conducted2-7. Other studies indicated that HPV is an independent risk factor for oral squamous cell carcinoma 8.

The advances in the area of genetics and molecular biology have decisively contributed to the study of the virus. Of all detecting HPV DNA techniques, polymerase chain reaction (PCR) is the most sensitive one.

Over 100 types of HPV have been identified to present. Out of these, 24 types were associated with oral lesions (HPV-1, 2, 3, 4, 6, 7, 10, 11, 13, 16, 18, 30, 31, 32, 33, 35, 45, 52, 55, 57, 59, 69, 72 and 73)9-11. The most prevalent type of HPV both in oral and genital lesions is HPV162.

The study of the involvement of HPV in the onset and progression of oral neoplasm has generated conflicting results. The discrepancy observed is attributed mainly to variation of sensitivity in the methods used and epidemiological factors of the examined groups 2. In our study, we conducted a bibliographic review about the oncogenic activities of HPV, as well as its correlation with carcinoma and benign lesions of oral epithelium, in order to contribute to the specialized literature on this controversial topic.

MATERIAL AND METHOD

Identification and selection of studies
Based on MEDLINE database, we studied medical literature articles written in English from January 1990 to December 2002, which referred to a correlation between HPV and normal oral mucosa, intraepithelial neoplasm (dysplasia and in situ carcinoma), verrucous carcinoma or oral squamous cell carcinoma. At MEDLINE, the keys words human papillomavirus, oral cancer, head and neck cancer, oral carcinoma, squamous cell carcinoma and oral lesions, were used separately and in combination. Based on the analysis of the list of references of ninety-six relevant publications and review articles, we selected forty-four papers published between January 1991 and December 2002. We also included six classical studies about HPV published in years previous to those of the study. The studies addressed immunological analysis, microscopy and molecular study to detect HPV in tissues or glands derived from normal oral mucosa, potentially malignant mucosa lesions and malignant oral lesions. We excluded data based on tissues that were not squamous, such as for example, salivary gland tumor, those resulting from distal sites of the anterior tonsil pillars, and those that had insufficient information to ensure definite results.

HUMAN PAPILLOMAVIRUS
Papillomaviruses are epitheliotrophic ubiquitous DNA viruses that infect the skin and mucosal epithelium, producing different types of benign and malignant epithelial neoplasms in animals and humans 12. They are associated with a rare variety of oral lesions and recently, we are highly suspicion that they can be implied in pre-malignant and malignant oral lesions. It is a 55nm diameter virus and it contains protein and a single spiral circular molecule of two DNA strands. The icosahedra particles of papillomavirus contain 72 capsomers. They depend on the terminal differentiation means of keratinocytes for replication, synthesis of capsid and virus assembly 12. It cannot be sufficiently cultivated in tissue cultures or animal models13.

The genome of papillomavirus can be divided into three regions: one long control region (LCR), comprising about 10% of the genome and early (E) and late (L) regions. The alignment of the sequences of HPV DNA reveals the genetic organization of the region that codifies viral proteins - open reading frame (ORF). They are present in one DNA strand and its functions were defined by comparing to the structure of type 1 bovine papillomavirus, which was extensively characterized in cell lines of genetically transformed rats. In general, regions E are expressed right after the infection and codify the proteins involved in inducing and regulating the synthesis of DNA. Conversely, regions L are expressed in late stages of infections and codify the proteins of the viral capsid. Regions R are designated E1 to E7 and regions L are divided into regions L1 and L2. From cell transformation perspective, regions E5, E6 and E7 are the most important ones 14.

The sequence between the end of L1 and the beginning of E6 is called long control region (LCR) and its is known also as the non-coding region (NCR). It contains many regulating cis sequences that control transcription and replication. There is substantial evidence that the progression of cancer induced by HPV is a multi-staged process 15. It has been advocated that LCR is a direct target for an intra-cell regulating mechanism 16. The progression to malignancy is followed by loss of intra-cell mechanism of inspection, which is correlated with abundant viral gene expression 17.

HPV infection starts when a viral particle penetrates the basal cells and undifferentiated and dividing cells of the epithelium. Upon a minor trauma, during sexual intercourse, it allows the virus to penetrate the basal layer of the epithelium. In basal and parabasal cells, the viral DNA replicates at low grade and only early genes are transcribed, also at low grade. Extensive multiplication of viral DNA and transcription of all viral genes, as well as formation of capsid, happen only on the more superficial layers of the epithelium. The virus multiplies exclusively in the nucleus of the infected cells. However, pathological manifestation associated with HPV is confined to sites in which the infection started 10.

Mature viral particles (with complete capsids) are, therefore, absent in basal cells and productive replication of HPV is restricted to cells in spinosum and granulosum strata 18.

According to the potential risk of development of malignant neoplasia in humans, the International Agency of Cancer Research in 1997 classified HPV 16 and 18 as human carcinogenic viruses (group 1), HPV 31 and 33 as probably human carcinogenic (group 2A) and some remaining types of HPV as possibly human carcinogenic (group 2B)19, 20. Another classification concerns the potential to malignancy and classifies the types as low (6, 11), intermediate (31, 33, 35) or high risk of malignancy (16, 18)21. They can also be classified according to anatomical site of infection and/or philogenetic analysis in mucous HPV or cutaneous HPV 22.

It was suggested that HPV is ubiquitous and that the common finding of the same types of HPV (6, 11, 156 and 18) in genital and oral mucosa are strong evidence of orogenital transmission 5. This virus has high tropism for mucosal epithelium, and it can be acquired by sexual transmission 10, 23. Most of the infections by HPV are a product of self-inoculation of the genital or oral site to another one 24. It can also be transmitted early at birth from the genital tract of the mother to the oral cavity of the child6, 20.

HPV IN NORMAL ORAL MUCOSA
A wide variation of the incidence of HPV infection detected in normal oral mucosa of healthy subjects has been reported, ranging from 0% to 81.1% in many studies, using different methods and a limited number of subjects. High prevalence (81.1%) was detected in the normal oral cavity of subjects using the highly sensitive method of PCR. It was suggested that the prevalence of HPV included subclinical infections and/or latent infections and that the infection with fewer copies of the virus was common in the oral cavity10. Surprisingly, HPV 18 was the most frequent genotype in normal oral mucosa 10, 25. HPV 18 infections in the oral cavity without lesions suggest that it persistently or frequently infects the oral mucosa, which can act as a reservoir of HPV. Regardless, it is still undefined whether HPV18 infection is subclinical and/or latent and persistent and/or transient 13.

Other studies, however, showed greater prevalence of HPV 16 in normal oral mucosa, present in symptomatic or latent form 3. In a wide review of literature, HPV 6, 11, 16 and 18 were the most prevalent in normal oral mucosa 26. Since it was clear that HPV infection of the normal oral mucosa was common, pathogenetic functions of HPV in oral cancer remained undefined and limited27.

HPV AND CARCINOGENESIS

The importance of HPV infection in oral carcinogenesis is supported by the capacity of high risk HPV of immortalizing oral keratinocytes in vitro20. Immortalizing can involve deactivation of suppressing proteins of pre-formed tumors by viral oncoproteins, block of suppressing gene transcription of tumors as a result of the insertion of HPV oncogenesis or the stimulation of cell oncogenesis transcription by inserting activating sequences of transcription derived from HPV. Thus, the infection of oral keratinocytes with high risk HPV can be involved in the pathogenesis of some oral squamous cell carcinomas, despite the evidence that implies HPV in the oral carcinogenesis is to present only circumstantial 28. Prevalence of HPV in oral cancer has ranged from 0% to 100% in literature reports, especially owing to variation in size of the sample, studied population and sensitivity of the employed techniques 10, 29. However, in genital lesions, such as condilomas, intraepithelial neoplasm and cervical squamous cell carcinoma, HPV has been recognized as a definite causal agent3.

Protein E7 of HPV-16 is capable of binding to pRb, which regulates the transition G1/S of the cell cycle, sequestering it, a possible mechanism through which HPV could contribute to carcinogenesis. Complex E7-pRb is detected in transformed human keratinocytes, despite the fact that it is not considered essential for the immortalization of these cells. Proteins E7 of high risk HPV form high affinity complex with many different cell proteins from the host, including pRb, whereas those low risk HPVs bind with low affinity 30.

Protein E7 binds preferably to hypophospholyrated pRb. As a result, it activates the transcription factor E2F, which is released from pRb. Such factors induce the transcription of important genes in the control of cell division, because they promote progression of the cell cycle, acting on steps G1 and S 31.

Protein E6 of HPV also shows an important role in cell transformation thanks to the capacity of forming complexes with p53, which protects the integrity of the cell genome. Those oncoproteins of HPV types 16 and 18 have shown not only capacity to form this complex, but also of degrading p53, through ubiquitin-dependent route 6.

Protein E6 of HPV is a polypeptide of approximately 150 amino acids, with apparent molecular weight of 18kD22. This protein has a half-life of 30 minutes to 4 hours in transformed cells. The oncogenic activities have been shown in many analyses. They include immortalization of the primary cells, transformation of defined cell lines, resistance to terminal differentiation, tumor genesis and cancellation of the checking point of the cell cycle 5, 33.

Once defined the connection, protein E6 stimulates the degradation of p53, which leads to very low levels in many human tumors, reason why cells in such tumors fail to interrupt the cell cycle G1, following the damage to cell DNA 31.

Since protein E6 of HPV 16 binds and degrades p53, it is intuitive to propose that it would suppress apoptosis. In fact, many studies in past years associating protein E6 with this cell event have been conducted and it is observed that there is an interference of the protein in different cell lines and as response to different inducing agents 33.

Recent evidence suggested that gene E5 of HPV 16 could also induce transformation in epithelial cells, possibly increasing transduction of intracell signal mediated by growth factors. Thus, genes E5, E6 and E7 of HPV would induce cell transformation and be involved in carcinogenesis 7, 13, 33.

Even after the publication of so many research studies on the topic, there is still a lot to be discovered, despite the possible hypothesis that carcinogenesis is a synergetic interaction of chemical, viral carcinogen elements, oncogenes and tumor suppressing genes 13, 33, 34.

The integration of viral DNA is also a genetic damage and the chromosome location of HPV integration has been mapped. Such viral contributions are early events in the development of cancer and viral integration can be an indicator of poor prognosis. The type of genetic damage can occur in different forms, such as gene amplification, chromosome translocation and loss of heterozygous (LOH) in HPV integration 35.

In most studied tumors and cell lines, the process of DNA integration in the host cell presents a cleavage of viral DNA at some point, located between genes E1 and E2 36 or between E1 and L1 37. When there is such separation, it seems to have an immediate consequence of interrupting the transcriptional control exerted by the translation unit (ORF) E2 over genes of the early region 36. It would lead to increased expression of proteins E6 and E7, and to uncontrolled cell proliferation and installation of a neoplastic process. HPV genome replicates as an episome in benign and pre-invasive lesions but it is integrated to the cell DNA of most cancers 38. However, integration of HPV in DNA of the host is not common in oral cancer3.

ASSESSMENT OF HPV DETECTION METHODS

Direct detection of HPV genomes and their transcripts can be obtained with procedures that include immunoperoxidase, hybridization Southern blot, Northern blot, dot blot and in situ, PCR, hybrid caption and DNA sequencing, among others 13.

Sensitivity and specificity of the various HPV detection methods available today vary widely. There are three categories that assess sensitivity of the methods: low sensitivity (immunoperoxidase, immunofluorescence and in situ hybridization) since they only detect virus if present in more than 10 copies of viral DNA per cell; the ones considered of moderate sensitivity (Southern blot, dot blot and reverse dot hybridization) since they detect virus only when there are 1 to 10 copies of viral DNA, and high sensitivity (PCR), when they detect virus in less than one copy of viral DNA present 25, 39. It is very important to consider within which context the method is being used. Southern blot hybridization is considered as the standard analysis for HPV genome and requires total length DNA fragments. It is a very valuable method nowadays because it provides important additional information, such as viral integration and subtyping 40.

In situ hybridization is a method employed today in large scale, which can be used with paraffin material, but when used alone, without PCR, it is not capable of detecting the virus when present in low number of copies of viral genome 3. PCR can be considered the appropriate technique in which small segments of DNA are normally expected, being considered a method that provides great sensitivity 40. It can amplify HPV genomes that result in exponential and reproducible increase of sequences of nucleic acid present in biological specimen. Under experimental conditions, it is the most sensitive detection method 41. Such methods should ensure quality, purity and in case of PCR we should avoid contamination. Tissues that have been fixated and soaked in paraffin can also cause problems because of duration of fixation and type of fixator used, which can affect considerably the quality of extracted nucleic acid. Upon selecting the appropriate method, such factors should be considered13.

In order to analyze HPV, Southern blot hybridization and DNA sequencing are procedures with excellent quality, but they take much time and money and can require great amounts of purified high quality DNA. Recently, the two most widely used methods that have equivalence in sensitivity are hybrid caption and PCR with general primers. This type of PCR is potentially capable of detecting all mucous HPV. Extensively applied generic protocols of PCR use one of the pairs of primers consensus GP5+/GP6+ and primers degenerated MY09/1142. The total differentiation of over 40 types can be managed by dot blot hybridization applying multiple type-specific probes 43 or restriction fragment length polymorphism (RFLP)42.

The assessment of efficacy of different techniques for detection of HPV is important and essential for the definition of HPV etiological role in oral lesions. Thus, a continuous analysis of new methods is essential to interpret the natural history of HPV infection in the oral cavity 13. Wide spectrum tests for HPV, easy to conduct and affordable, are expected and they will play an important role in molecular diagnostic systems.

DISCUSSION

As observed, studies developed to present do not allow us to precisely define which is the possible role of HPV in oral carcinogenesis, however, in cervical cases, the role is clearly defined13. Among the factors that generate controversy, we can highlight the frequent finding of various types of HPV in normal oral mucosa, an incidence that varies from 0% to 81.1%, which was detected in a study in the oral cavity of healthy adults using a highly sensitive method, PCR 10. It may be associated with subclinical and/or latent infections and an infectious clinical picture with few copies of the virus. What contributes to increasing the controversy is that in many studies, the most commonly found types are HPV 18, 16, 11 and 6, certainly associated with malignant and benign neoplasm, being the two first considered of high risk and the other two, of low risk 3, 13, 26. Since it became clear that normal oral mucosa infection by HPV was common, pathogenic functions of this virus in oral cancer remained undefined and limited 27. It is not known for sure whether the infection found is only a latent stage or if the oral cavity works as a HPV reservoir13.

The wide discrepancy in incidence of the virus in malignant and potentially malignant lesions found in many literature reports (ranging from 0% to 100%)2, 10, 29 has also contributed to the doubt, but the discrepancy is normally attributed to variation in sensitivity of the methods used, plus the diversification of the studied populations and the size of the sample 3. Studies that employed low sensitivity methods (immunoperoxidase, immunofluorescence, and in situ hybridization) did not detect the virus or had very low positive levels of the virus 25, 29. For this reason, the use of high sensitivity methods is a fundamental condition for the conduction of high reliability tests. In special, the technique that gathers the highest levels of sensitivity and specificity is the association of PCR and Southern blot hybridization because it allows identification and precise typification of the virus. As to the studied population, it has been suggested that the prevalence of infection is higher in oral lesions of patients in India and lower in Western countries (North America and Brazil)3, which influences significantly the results, in addition to the fact that the larger the sample employed in the study, the more reliable the results. Another aspect to be highlighted is the existence of other carcinogenic factors that act over the recovering epithelium of the oral mucosa and, among them, smoking and alcohol have an important role 44. Owing to power and frequency, they have oncogenic confirmed action over the oral epithelium, which hinders the study on the action of HPV, by coexisting with the other two agents that are as important as the one studied here.

Conversely, the role of high risk HPV in oral cancer has been increasingly perceived as more evident. Most of the published studies involving the role of the virus in the process in different countries has shown a minimum positive index of 20% 20. In addition, the immortalization of oral keratinocytes in vitro by high risk HPV has been confirmed by many researchers, ratifying the importance of the virus as a carcinogenic biological agent 13, 20, 28. Regardless, the role of proteins E5, E6 and E7 of high risk HPV has been increasingly understood, emphasizing the participation of the virus in the carcinogenic process, since it induces cell transformation 7, 13, 33.

FINAL REMARKS

In view of the facts reported here, it is evident that the participation of HPV in oral cancer is associated with a part of the oral carcinomas and that its action in the process is synergic, that is, associated with other chemical and physical carcinogenic factors of relevance, such as smoking and alcohol use. Only a small percentage of oral carcinomas would be associated exclusively with the action of viral proteins E5, E6 and E7, without the effective participation of the previously referred chemical and physical carcinogenic elements. Effective detection methods and preventive measures are absolutely essential in the fight against the virus and the search for evidence of its participation in oral cancers, which are common topics for studies that researchers are currently conducting and will in the future.

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1 Master degree and Doctorate studies in oral Pathology under course - UFRN; Assistant Professor, Course of Dental Sciences, UEFS.
2 Master in Genetics and Molecular Biology, Doctorate studies in Oral Pathology under course - UFRN.
3 Ph.D., Professor, Program of Post-Graduation in Oral Pathology - UFRN.
4 Ph.D., Professor, Program of Post-Graduation in Oral Pathology - UFRN.
Program of Post-Graduation in Oral Pathology - UFRN (Federal University of Rio Grande do Norte)
Address correspondence to: Prof. Dr. Antônio de Lisboa Lopes Costa - Av. Industrial João Motta, 1541 Bl. B, Ap. 301 Capim Macio Natal RN 59082-410
E-mail: antoniodelisboa@uol.com.br
Article submitted on January 27, 2003. Article accepted on July 10, 2003.