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Microbes and Infection xx (2014) 1e8 www.elsevier.com/locate/micinf
Review
Viruses and oral cancer. Is there a link? Lars Sand*, Jamshid Jalouli Department of Surgical Sciences, Oral and Maxillofacial Surgery, Medical Faculty, Uppsala University, Akademiska sjukhuset, 751 85 Uppsala, Sweden Received 22 November 2013; accepted 24 February 2014
Abstract Oral squamous cell carcinoma (OSCC) is the most common malignant tumour of the oral cavity. The aetiology of epithelial cancer of the head and neck is considered to be a multifactorial, sequential process. DNA viruses are found in many different cancers and are also capable of transforming cells to a malignant phenotype. Human Papilloma Virus (HPV) has been proposed as risk factors in OSCC development and HPV type 16 is the most important subtype. Other oncogenic virus species i.e., EpsteineBarr Virus and Herpes Simplex Virus Type 1 have been proposed to be involved in oral carcinogenesis. However, no convincing evidence exist that they are an established risk factor in OSCC. Therefore more studies are needed in order to clarify the different aspects of virus involvement. Here, we review the existing literature on viral involvement in oral cancer. Ó 2014 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.
Keywords: Oral cancer; Human Papilloma Virus; EpsteineBarr Virus; Human herpes simplex virus type 1
1. Introduction Oral squamous cell carcinoma (OSCC) is the most common epithelial malignancy of the oral cavity. OSCCs and their variants constitute over 90% of oral malignancies, and the disease is associated with poor prognosis. OSCC is a complex malignancy where environmental factors, viral infections, and genetic alterations most likely interact, and thus give rise to the malignant condition. Viral causes of cancer have been studied since the beginning of the 20th century, when an infective agent, which later was shown to be a virus that had the ability to induce tumours in chickens, was isolated [1,2]. During the 1930s, Richard Shope was able to transfer a papillomavirus, by using cell-free medium, to rabbits [3]. Groupe et al. showed in various studies that leukaemia and sarcoma could be induced in mice through an infectious virus [4]. Another major step was made when two research groups successfully transformed cells which were infected with Rous sarcoma virus, into mice [4,5]. It was * Corresponding author. Tel.: þ46 18 6116450. E-mail addresses: [email protected], [email protected] (L. Sand).
thereby possible to identify specific viral oncogenes that could transform normal cells into cancer cells. Viral involvement in the development of OSCC has been proposed in many studies. The most frequently studied virus species are Human Papilloma Virus (HPV), EpsteineBarr Virus and Herpes Simplex Virus Type 1 (HSV-1). Some authors have confirmed a clear connection, while other authors have not been able to find any convincing evidence. Coinfection by two or more virus species has been suggested as an increased risk factor for cancer development in general [6], but also in OSCC [7e10]. There now seems to be a consensus that HPV has oncogenic capacity not only in cervix cancer but also in oral, as well as head and neck cancer. Regarding the other oncogenic viruses, the situation is more doubtful. In this article, we review available literature regarding viral involvement in the development of OSCC. 2. Oral cancer Oral cancer is usually defined as a neoplastic disorder in the oral cavity, which includes the following areas: lip, buccal
http://dx.doi.org/10.1016/j.micinf.2014.02.009 1286-4579/Ó 2014 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved. Please cite this article in press as: Sand L, Jalouli J, Viruses and oral cancer. Is there a link?, Microbes and Infection (2014), http://dx.doi.org/10.1016/ j.micinf.2014.02.009
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mucosa, lower and upper alveolar ridges, retromolar gingiva, floor of the mouth, hard palate, and the anterior two-thirds of the tongue. By gross morphologic examination, one sees exophytic, ulcerative, or verrucous types. Squamous cell carcinoma (SCC) constitute 90% of epithelial malignancy in the oral cavity and over 50% of the tumours have an apparent “precancerous” state, often preceded by potentially malignant disorders such as leukoplakia, oral lichen planus and oral submucous fibrosis [11e13]. The aetiology of epithelial cancer of the head and neck is considered to be a multifactorial, sequential process. The continuum of SCC progress from individual epithelial cell changes (atypia) to a generalized disturbance of the epithelium (dysplasia), and then to carcinoma in situ, and finally to invasive SCC. The malignant tumour, consisting of strands of malignant epithelial cells infiltrating subepithelially, may resemble any or all of the layers of stratified squamous cell epithelium. The two major aetiological factors in SCC of the oral cavity are the social habits of tobacco use and alcohol consumption [14,15]. Different groups of genes are involved in the multiple genetic events of malignant cell transformation: oncogenes, tumour-suppressor genes, DNA repair genes, and DNA sequences that control apoptosis. The normal function of the p53 tumour-suppressor gene, located on the short arm of chromosome 17, is that of “guardian of the genome”. Damage to DNA is associated with nuclear accumulation of the p53 protein, presumably inducing growth arrest for repair or the induction of apoptotic cell death [16]. Mutation in the p53 gene is frequently found in human cancer, and also in SCC of the head and neck. These mutations likely result from carcinogen-induced DNA damage. In patients with head and neck SCC, using high amount of tabacco and alcohol, elevated p53 expression has been detected [17,18]. Elevated p53 has also been associated with HPV-infection [19]. OSCC is treated with surgery, radiotherapy, chemotherapy or a combination of these three modalities. The main prognostic factors are tumour size/stage, presence of locoregional metastasis and sub site and the 5-year survival rate is approximately 50% and has not improved much over the last decades. HPV-infected OSCC, however, seems to have a slightly better prognosis [20]. 3. Oncogenic viruses and oral cancer 3.1. Human Papilloma Virus (HPV) 3.1.1. Historical background Papillomavirus infection in the oral mucosa was first demonstrated in animals by DeMonbreun et al., in 1932 [21]. Yet not until 1967 did Frithiof et al. present the first ultra structural evidence of the presence of papillomavirus in human oral papillomas, and Praetorius-Clausen et al., in 1971, demonstrated particles compatible with papovavirus in oral focal epithelial hyperplasia [22]. Jenson et al. were first to detect HPV antigen in oral verrucae, multiple papillomas, and condylomata [23]. Later, after the development of
hybridization techniques, especially the very sensitive polymerase chain reaction (PCR), numerous investigations have detected different types of HPV in different oral lesions, as well as in normal oral mucosa. 3.1.2. HPV characteristics The papillomaviruses, which replicate in the nucleus of squamous epithelial cells, belong to the papovavirus group, and are small, non-enveloped DNA viruses of a symmetrical icosahedral shape. Papillomavirus particles (52e55 nm in diameter) consist of a single molecule of double-stranded, circular DNA with approximately 8000 bp, contained in a capsid (spherical protein coat) composed of 72 capsomeres (repeating subunits of the capsid) [24]. In a virus, only the genome is present, with no cellular machinery for replicating the genome or manufacturing the capsid. The host cell must supply the necessary ingredients for the assembly of the viral components. When cellular death does not occur during viral reproduction, chronic infection can probably succeed. HPV is a very large and heterogenous group of DNA viruses and over 100 types have already been identified. Based on the clinical behaviour of HPV infections, HPV viruses can be grouped into high-risk (HR) and low-risk (LR) HPV types. HR-HPVs are associated with lesions that have a propensity to undergo carcinogenesis, and these viruses include types 16, 18, 31, 33, 35, 39, 45, and 52. 3.1.3. Carcinogenesis of HPV Initially, HPV infects undifferentiated proliferative basal cells, which are capable of dividing. Once inside the host cell, viral DNA localizes into the nucleus and establishes itself as an episome with a low copy number (some 10e200 copies per cell). At this stage, the viral proteins E1, E2, E6, and E7 transcribed from the early promotor are expressed at a low level [25]. Mainly E6 and E7 may disturb the normal terminal differentiation by stimulating cellular proliferation and DNA synthesis. After the onset of genome amplification, the capsid proteins L1 and L2 accumulate in the mature epithelial cells. The assembly of infectious virions takes place in terminally differentiated cells of the upper epithelial layers, and the virions are shed to the environment, as the cells are lost through desquamation. In addition to active replication, HPV infection can result in latency and malignant transformation through interactions of viral E6 and E7 proteins with p53 and pRB [26]. The tumour suppressor p53 can dictate cellular fate by initiating cell cycle arrest, promoting DNA repair, triggering apoptosis, and inducing growth arrest/senescence. Thus a direct mutation can alter or inactivate p53, and interactions with the oncogene products of HPV (e.g., E6) can also cause aberrations in p53 regulation [27]. The viral E7 protein binds and inactivates a human tumour suppressor gene product, the retinoblastoma protein (pRB) and the viral E6 protein binds p53 and earmarks it for destruction by the ubiquitin pathway. In malignant tumours, including SCC of the head and neck, somatic mutations in the p53 gene, or in other cellular genes that modulate p53 activities, commonly inactivate p53 function. Such E6 elimination of p53 represents a critical step in
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HPV-induced carcinogenesis, in that it provides an alternative way of disrupting the p53 tumour suppressor gene pathway in the cancer cell [28]. The state of the virus (i.e., whether it is maintained as an episome and not integrated, or is integrated into the host genome) seems to play an important role as well. During the normal HPV life cycle, viral DNA is maintained episomally in the nucleus of the affected cell, a state predominantly associated with “low-risk” HPV types such as HPV-6 and -11. 3.1.4. HPV and OSCC The relationship between cervical HPV infection and cervical cancer is well established, and in over 90% of cervical intraepithelial neoplasias (CIN) and cervical cancers, HPV is present [29]. Infection of the cervical epithelium with specific high-risk types of HPV plays a fundamental role in the development of cervical cancer, by causing precursor lesions and maintaining malignant growth [30]. Two review-articles indicated that a similar mechanism is seen in oral potentially malignant disorders [31,32]. Numerous studies have examined the relationship between HPV and SCC in the head and neck region (for review see Hillbertz et al., 2012) [33]. The clear association between HPV and cervical cancer is not as strong for OSCC. HPV integration into host DNA is less common in OSCC [34,35], and in a systematic review, Kreimer et al. found the prevalence of HPV to be 24% in OSCC [36]. Another study reported the estimated proportion of oral and oropharyngeal SCC attributable to HPV infection to be 35% [37]. Na¨sman et al. demonstrated an increasing incidence of HPV-positive tonsillar cancers in the Stockholm area, being 23% in 1970, 68% in 2002, and 93% in 2007 [38]. A strong causal association between HPV infection and tonsil-related cancers was also seen in Montreal [39]. There is also a growing incidence of HPV-associated OSCC in the USA [40], a trend which has been demonstrated in other developed countries [38,41,42]. A prospective study has found that increased risk for head and neck cancer was observed more than 15 years after HPV exposure. Men generally have a higher incidence of oral HPV infection than women but the degree to which tobacco and/or alcohol use increase the risk of HPV-positive OSCC is unclear [43]. However, HPV-positive OSCC may be the main cause of OSCC in non-smokers and non-drinkers. Poor oral health is an additional risk factor for HPV infection, irrespective of smoking and oral sex practice [44]. Further, immunosuppression seems to be an increased risk factor for HPV-positive OSCC [45]. Of the numerous HPV viruses, zur Hausen demonstrated that HPV-16 plays a dominant role in precancerous and cancerous lesions of the cervix, followed by HPV-18 [46]. A similar pattern is seen in OSCC where in most studies, HPV types 6, 11, 16, and 18 have been found, with a clear dominance of HPV type 16 [47e49]. There seem to be enough evidence to claim that HPV is an independent risk factor for OSCC development. It is estimated that 25e35% of all OSCC are caused by HPV.
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3.2. EpsteineBarr Virus 3.2.1. Historical background Dennis Burkitt, a missionary doctor in East Africa in the 1950s, was the first to recognize and describe Burkitt’s lymphoma [50,51]. After a meeting in London in 1961, Dr. Burkitt met Tony Epstein, who together with Yvonne Barr managed to establish Burkitt’s lymphoma-derived cell lines in culture [52]. Examining these cell lines with the electron microscope provided clear evidence of herpes-like particles in a small portion of cells [53]. This virus, distinct from other members of the human herpes virus family, was the first human tumour virus identified. The virus was named the EpsteineBarr Virus. 3.2.2. EBV characteristics The human herpes virus family consists of three subfamilies, i.e., alpha, beta, and gamma. EBV belongs to the gamma subfamily, which is split into two genera, lymphocryptovirus and rhadinovirus. EBV is the prototype for the lymphocryptovirus because it latently infects B-lymphocytes [54]. Like other herpes viruses, EBV has a toroid-shaped protein core, wrapped with DNA; a nucleocapsid with 162 capsomeres; a protein tegument between the nucleocapsid and the envelope; and an outer envelope with external glycoprotein spikes. The EBV stores its genome in the form of a linear, double-stranded, 172 kbp DNA molecule [55]. Two EBV subtypes have been identified, i.e., EBV-1 and EBV-2, differing in the genes coding for nuclear proteins EBNA-LP, 2, -3B, and -3C, with differences in the predicted amino acid sequence of between 28% and 47% [56]. 3.2.3. Carcinogenesis of EBV EBV is one of the most common and widespread human viruses, infecting over 95% of people worldwide. After acute infection with or without symptoms, latent EBV infection will persist lifelong. Hence, EBV has the ability to establish a latent infection, which means a silent state of viral infection characterized by the low expression of viral genes and minimal cytopathic effects or production of infectious virus. EpsteineBarr Virus has been identified as the causative agent of infectious mononucleosis. EBV enters the host through the oropharynx and has developed a way to simultaneously access both B cells and squamous epithelial cells of the oropharynx. However, B cells are considered necessary and sufficient for EBV infection, while epithelial cells may be seen as helpful enhancers for transferring the virus to others, and for establishing latency in B cells [57]. In most cases, transmission occurs orally via the saliva. EBV gene products synthesized in latent infections are of particular interest in relation to the oncogenic properties of this virus. A number of gene products have been characterized that are expressed in specific tumours or in non-transformed, latently infected cells. Some of these share structural and functional homologies with cellular gene products. The nuclear EBNA-1 protein is consistently expressed; this is a sequence-specific DNA-binding phosphoprotein that plays a central role in the episomal maintenance of the EBV genome and is required for its DNA replication [58].
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EBV is associated with “oral hairy leukoplakia”, which is a non-malignant white lesion at the lateral boarder of the tongue in immunodeficient individuals [59]. Burkitt’s lymphoma (BL), a non-Hodgkin, monoclonal Blymphocyte tumour of low differentiation grade, frequently presents in the jaw, and is found endemically in equatorial Africa and Papua New Guinea. Almost 50% of the affected patients are children. More than 90% of the BL in these areas contain EBV genomes. Outside these areas, only sporadic cases are seen. In sporadic BL, EBV can be detected in only 10e20% of the tumours [60]. Nasopharyngeal carcinoma (NPC), an epithelial cell tumour that develops in the space behind the nose that is rich in lymphocytes is endemic in people from Southern China; native North Americans; Tunisians; and East Africans. In Cantonese Chinese and native North Americans, 20% of all cancers may be NPC, whereas in Western countries, less than 1% of all cancers are NPC [60]. In contrast to BL, EBV is present in every anaplastic NPC cell. Genetic predisposition, environmental factors, and nutritional factors are thought to be important in NPC aetiology [61]. 3.2.4. EBV and OSCC In vivo, EBV infects two types of target cells in the oral region: epithelial cells in the oro/nasopharynx and/or salivary gland, and the B-lymphocytes [62]. Thus, this has been the reason for the suspicion that EBV is involved in OSCC and premalignant lesions. Using PCR, variability in EBV prevalence has been high in previous studies of OSCC, premalignant oral lesions, and normal oral mucosa [63,64]. EBV has been associated with OSCC in some studies [8,65,66], while in other studies no clear association between EBV and OSCC has been found [67,68]. In 145 OSCC samples from toombak users in Sudan, EBV was detected in 53 (37%) of the samples. The corresponding figure for the OSCC samples from nontoombak users was 16 (22%) for EBV [10]. EBV DNA was detected in 18 (29%), from 62 Indian patients with betelassociated OSCC [9]. This association between smokeless tobacco and EBV was not found in a study from Sweden on patients with snuff-induced lesions [64], and even though Sand et al. found a significant increase in EBV prevalence in OSCC compared to healthy controls, no correlation with tobacco use was seen [65]. While some authors found a clear difference in EBV positivity between oral lesions and controls, others found higher EBV prevalence in the controls. This high variability and inconsistency probably reflects differences in ethnicity, geographical differences as well as differences in methodological sensitivity [9,10]. The variable EBV prevalence in OSCC in different studies might be explained by different sampling techniques, divergent PCR methods and also the quality of the sample (e.g. whether frozen or fixed). The prevalence of EBV in oral samples varies widely between ethnic and geographical populations. In a study by Jalouli et al., 85 (55%) of 155 examined OSCCs from 8 different countries were EBV-positive, but the variation between countries was large (22e80%) [63]. Southeast Asian studies
found a high prevalence of EBV and suggested an aetiological role of EBV in OSCC [69]. Through immunohistochemical detection of LMP-1, the EBV antigen was associated with transforming cell activity in an Egyptian population [70]. By contrast, North American studies, as well as West and North European studies, regularly report lower prevalence of EBV [67]. Even though there are plenty of reports showing an association between EBV and OSCC development, no safe conclusions could be drawn from those. Further, many reports show no association. More and larger population based studies are needed to prove the oncogenic potential of EBV in OSCC. 3.3. Herpes Simplex Virus Type 1 (HSV-1) 3.3.1. Historical background The clinical manifestation of oral herpetic infections has been known for 250 years, but the causative agent was not identifies as a herpes virus until the 20th century. In 1962, Schneweis identified two serotypes [71], subsequently confirmed by Nahmias and Dowdle [72]. A casual association between a herpes virus and renal adenocarcinoma of the leopard frog was suggested by Lucke´ in 1938 [73]. Additional experimental studies seemed to support a possible role of HSV in human cancers. These originated in part from reports of HSV-DNA or RNA persistence in cervical cancer cells: in 1972, Frenkel et al. and in 1973, Roizman and Frenkel, identified a fragment of HSV-2 in one cervical cancer biopsy and also analysed transcripts of this DNA [74,75]. 3.3.2. HSV-1 characteristics Herpes simplex virus belongs to a family of eight related viruses, including herpes simplex virus types 1 (HSV-1) and 2 (HSV-2), varicella-zoster virus, EpsteineBarr Virus, and cytomegalovirus. Like all herpes viruses, HSV-1 and the closely related HSV-2 have enveloped, spherical virions. The genome of HSV-1 is densely packaged in a liquid-crystalline, phage-like manner in a 100-nm icosahedral capsid [76]. The genome of HSV-1 consists of linear double-stranded DNA, with approximately 150 000 bp. A significant property of all herpes simplex viruses is their ability to remain latent in host neurons for life, and can reactivate to cause lesions at or near the site of initial infection. 3.3.3. Carcinogenesis of HSV-1 Infection with herpes simplex viruses (HSV) is very common worldwide. In European countries, the estimated prevalence of HSV-1, determined by means of antibody measurements, varies between 60% and 90% in the general population [77]. The prevalence of HSV-1 exceeds 40% by age 15 years and rises to 60e90% in older adults. In the USA, there are estimated to be approximately 500 000 primary infections per annum [78]. After inoculation of the skin or mucous membrane, HSV-1 is transported along sensory axons in a retrograde direction to the neuronal cell body, where it establishes lifelong latent infection. Periodic reactivation results in HSV-1 being transported in an anterograde direction to
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nerve terminals, where it causes recurrent clinical disease or asymptomatic viral shedding [79]. HSV-1 infection is primarily oro-pharyngeal and HSV-2 infection is primarily genital, even though HSV-1 has been found in genital lesions and HSV-2 in oral lesions. Facial herpes may establish latency in the trigeminal ganglion, while genital herpes may involve the sacral ganglion. Herpes simplex infection generally occurs in two phases: the initial, primary infection, followed by secondary, recurrent disease at the same site. In the first phase, the virus spreads by close person-to-person contact with lesions or mucosal secretions (e.g., saliva or cervical discharge) as well as via respiratory droplets. The incubation period is 2e10 days; the virus then spreads to regional lymph nodes, causing tender lymphadenopathy [80]. Though HSV-1 infections are frequently asymptomatic, they can produce a variety of signs and symptoms. These include oral or perioral lesions, ocular infections, congenital skin lesions, genital skin or mucous membrane lesions, and serious systemic illnesses such as encephalitis and neonatal disease. One of the major features of HSV infection is the ability of the virus to remain latent in the sensory ganglia. Reactivation from the latent state can be triggered by external stimulus and results in axonal transportation of the virus back to the epithelium, leading to either clinical lesions with lytic (productive) destruction of distal epithelial cells or subclinical shedding. Increased antibody levels against HSV-2 have been reported on women with cervical cancer. A large-scale seroepidemiological study by Smith et al. linked HSV-2 seropositivity with increased risk of cervical squamous cell carcinoma [81]. On the other hand, a Scandinavian study on 550 000 women come to the opposite conclusion [82]. Integration of HSV DNA in tumours would be an indication of HSV involvement in carcinogenesis. There have been reports on HSV DNA and RNA in cervical tumours, while several studies have not been able to detect any HSV DNA or RNA in malignant cervical lesions. However, several studies have shown regions of homology between HSV and human DNA, which make the interpretation of these studies difficult. A “hitand-run” mechanism has also been proposed for HSV oncogenesis [83]. 3.3.4. HSV-1 and OSCC The role of HSV in the development of OSCC has been investigated in both animal models and in vivo. In the first animal model on this topic, Southam et al. demonstrated that HSV could be a co-carcinogen. They showed that an infection with HSV-1 could increase the proportion of animals with carcinomas from 7 to 19% [84]. Subsequently, Duff and Rapp showed that if the virus was partially inactivated with various agents to prevent it from killing cells, a certain number of the surviving cells would then become transformed to a malignant phenotype. These transformed cells did not only invade local tissue but also metastasize to distant sites in the animals [85]. In a study of hamsters, Stich et al. demonstrated that snuff significantly enhanced herpes simplex virus-associated development of micro-invasive SCC in cheek pouch epithelium
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[86]. In a study of rats, Larsson et al. demonstrated that HSV-1 and snuff interact in the development of malignant lesions [87], and Hirsch et al. demonstrated that the incidence of malignant tumours was significantly higher in rats exposed to snuff and HSV/snuff than in control animals. Further, exposure to both the tobacco and the virus produced more tumours than exposure to either agent alone. One explanation would be that HSV-1 potentiates tumours that are induced by carcinogens in the tobacco [88]. Cornella et al. used an animal model where hamsters were exposed to intraoral infections with HSV-1, and after recovery, the cheek pouches were exposed to the carcinogen 7,12dimethyl-benz[a]anthracene [DMBA]. The number of tumours was significantly higher in animals that were previously infection with HSV-1 [89]. A similar situation to humans would be where HSV-1 becomes latent in the trigeminal ganglion after which the oral mucosa is exposed to tobacco carcinogens. Clinical studies have shown a possible interaction between smokeless tobacco and HSV-1 in the development of OSCC [7]. Jalouli et al. investigated the presence of HSV-1 DNA in tobacco-related OSCC from a Sudanese and an Indian population [9,10]. In toombak-related OSCCs, HSV-1 was detected in 29% of the samples, compared to 24% in nontoombakrelated OSCCs and HSV-1 DNA was detected in 5% of the 62 patients with betel-related OSCC. In a subsequent study they also compared OSCCs from 8 different countries regarding HSV-1 [63]. The overall prevalence of HSV-1 positive OSCC was 15%. However, the difference between the countries was large, with the UK having the highest prevalence (55%). There was a statistically significant higher HSV-1 prevalence in the industrialized countries compared to the developing countries, mainly due to the high prevalence in the UK. No safe conclusions could be drawn from these studies but the ethnic composition and socio-economic situation seem to be important factors regarding the prevalence of HSV-1 in OSCC. Meurman claim in a review study from 2010, that HSV infections link statistically with oral carcinogenesis and that antibody levels to HSV-1 and HSV-2 are increased in OSCC patients [90]. A population-based study from USA, suggests that HSV-1 may enhance the development of OSCC [91]. However, another American study came to the opposite conclusion where the risk of OSCC was not significantly increased in those with HSV-1 or HSV-2 antibodies compared to HSV-negative patients [92]. Taken together, no evidence that HSV acts as a direct carcinogen in OSCC exists to date. The transformation studies were mostly performed 2e3 decades ago, and the results of recent studies are inconclusive and contradictory. 4. Viral detection methods Several methods for detection of viral DNA have been used, including PCR assays, in situ hybridization (ISH), immunohistochemical staining (IHC), dot blotting and southern blotting [93].
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Before the era of molecular techniques, serological tests were common for detection of EBV and HSV-1. For detection of HPV infection, serology is an unreliable method, because frequent cross-reactions and transient antibody responses reduce sensitivity and specificity, which therefore is low, especially when compared to PCR. Increased antibody titres against EBV in serological tests are strongly correlated with EBV induced malignancies such as NPC. EBV associated tumours are often characterized by early antigens and IgG viral capsid antigens with diminished EBNA titres. However, this pattern is not specific for malignancy, implying that serology alone is inadequate for diagnosing EBV-related malignancies. The main advantages of molecular techniques are its higher sensitivity and specificity. In situ hybridization (ISH), widely used for virus detection, can be utilized on formalin-fixed and paraffin-embedded sections from biopsy specimens. ISH has the advantage of providing information on whether HPV is present, and if so, where HPV DNA is located in the tissue. In addition, information about morphological abnormalities associated with HPV can then be provided. ISH, which can localize latent EBV in tissue samples, has been used for detection of EpsteineBarr encoded RNAs in investigations of the association between EBV and malignancies [94]. ISH has a disadvantage of low sensitivity, compared to PCR, with a detection limit of about 20 viruses per infected cell, and ISH is also more time consuming. Southern blot hybridization (SBH) is a process in which complementary single strands of nucleic acids combine to form a stable double-stranded nucleic acid molecule. The main disadvantages of SBH compared with PCR are low sensitivity, large amounts of cellular DNA required, and greater expense. However, despite these drawbacks, SBH is useful for confirming PCR results, especially in the case of paraffin-embedded tissue samples, in which background amplification bands may preclude interpretation of data from ethidium bromide staining. The polymerase chain reaction (PCR) assay is a technique that offers several advantages over other methods. It requires only a small quantity of biological material and can detect the viral presence in “early” infections. PCR detection of HPV, EBV, and HSV is highly sensitive and specific, and can supplement the detection of clinical manifestations of virusassociated oral lesions [95]. PCR and variants of PCR are currently the most commonly used viral detection method. The disadvantage however, is the risk of contamination and internal controls are of most importance in PCR-based assays. Additionally, several methods and newer techniques are constantly under investigation. 5. Conclusions There now seems to be a consensus that HPV has oncogenic capacity not only in cervical cancer but also in oral, as well as head and neck cancer. Review-studies and metaanalyses indicate that approximately 25e30% of oral cancer could be attributed to HPV and HPV type 16 is the most important subtype in oral carcinogenesis. Regarding the other
oncogenic viruses, the situation is more doubtful. At present, it would be fair to claim that the aetiological role of EBV in oral SCC is highly questionable. If HSV plays an active role in OSCC development or if it is only a passive by-stander in the decreased local immune-deficient tumour area, remains unclear. The fact that some studies indicate that co-infection by multiple oncogenic viruses may be important risk factors is OSCC development needs further investigation. PCRbased assays are currently the mostly used viral detection method. References [1] Ellerman C, Bang O. Experimentelle Leuka¨mie bei Hu¨hnern. Cent Bakteriol 1908;46:595e609. [2] Rous P. A sarcoma of the fowl transmissible by an agent separable from the tumor cells. J Exp Med 1911;13:397e411. [3] Shope R. Serial transmission of virus of infectious papillomatosis in domestic rabbits. Proc Soc Exp Biol Med 1932;32:830e2. [4] Groupe V, Manaker R. Discrete foci of altered chicken embryo cells associated with Rous sarcoma virus in tissue culture. Virology 1956;2:838e40. [5] Temin H, Rubin H. Characteristics of an assay for Rous sarcoma virus and Rous sarcoma cells in tissue culture. Virology 1958;6:669e88. [6] Yang YY, Koh LW, Tsai JH, Tsai CH, Wong EF, Lin SJ, et al. Correlation of viral factors with cervical cancer in Taiwan. J Microbiol Immunol Infect 2004;37:282e7. [7] Yang YY, Koh LW, Tsai JH, Tsai CH, Wong EF, Lin SJ, et al. Involvement of viral and chemical factors with oral cancer in Taiwan. Jpn J Clin Oncol 2004;34:176e83. [8] Al Moustafa AE, Chen D, Ghabreau L, Akil N. Association between human papillomavirus and Epstein-Barr virus infections in human oral carcinogenesis. Med Hypotheses 2009;73:184e6. [9] Jalouli J, Ibrahim SO, Mehrotra R, Jalouli MM, Sapkota D, Larsson PA, et al. Prevalence of viral (HPV, EBV, HSV) infections in oral submucous fibrosis and oral cancer from India. Acta Otolaryngol 2010;130:1306e11. [10] Jalouli J, Ibrahim SO, Sapkota D, Jalouli MM, Vasstrand EN, Hirsch JM, et al. Presence of human papilloma virus, herpes simplex virus and Epstein-Barr virus DNA in oral biopsies from Sudanese patients with regard to toombak use. J Oral Pathol Med 2010;39:599e604. [11] Schepman K, der Meij E, Smeele L, der Waal I. Concomitant leukoplakia in patients with oral squamous cell carcinoma. Oral Dis 1999;5:206e9. [12] Noonan VL, Kabani S. Diagnosis and management of suspicious lesions of the oral cavity. Otolaryngol Clin North Am 2005;38:21e35. [13] Gupta PC, Bhonsle RB, Murti PR, Daftary DK, Mehta FS, Pindborg JJ. An epidemiologic assessment of cancer risk in oral precancerous lesions in India with special reference to nodular leukoplakia. Cancer 1989;63:2247e52. [14] Proia NK, Paszkiewicz GM, Nasca MA, Franke GE, Pauly JL. Smoking and smokeless tobacco-associated human buccal cell mutations and their association with oral cancer e a review. Cancer Epidemiol Biomarkers Prev 2006;15:1061e77. [15] Hirsch JM, Wallstrom M, Carlsson AP, Sand L. Oral cancer in Swedish snuff dippers. Anticancer Res 2012;32:3327e30. [16] Koh J, Cho N, Kong G, Lee J, Yoon K. p53 mutations and human papillomavirus DNA in oral squamous cell carcinoma: correlation with apoptosis. Br J Cancer 1998;78:354e9. [17] Ibrahim S, Johannessen A, Idris A, Hirsch J, Vasstrand E, Magnusson B, et al. Immunohistochemical detection of p53 in non-malignant and malignant oral lesions associated with snuff dipping in the Sudan and Sweden. Int J Cancer 1996;68:749e53. [18] Robinson M, Sloan P, Shaw R. Refining the diagnosis of oropharyngeal squamous cell carcinoma using human papillomavirus testing. Oral Oncol 2010;46:492e6. [19] Dai M, Clifford GM, le Calvez F, Castellsague X, Snijders PJ, Pawlita M, et al. Human papillomavirus type 16 and TP53 mutation in oral cancer:
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Microbes and Infection xx (2014) 1e8 www.elsevier.com/locate/micinf
Review
Viruses and oral cancer. Is there a link? Lars Sand*, Jamshid Jalouli Department of Surgical Sciences, Oral and Maxillofacial Surgery, Medical Faculty, Uppsala University, Akademiska sjukhuset, 751 85 Uppsala, Sweden Received 22 November 2013; accepted 24 February 2014
Abstract Oral squamous cell carcinoma (OSCC) is the most common malignant tumour of the oral cavity. The aetiology of epithelial cancer of the head and neck is considered to be a multifactorial, sequential process. DNA viruses are found in many different cancers and are also capable of transforming cells to a malignant phenotype. Human Papilloma Virus (HPV) has been proposed as risk factors in OSCC development and HPV type 16 is the most important subtype. Other oncogenic virus species i.e., EpsteineBarr Virus and Herpes Simplex Virus Type 1 have been proposed to be involved in oral carcinogenesis. However, no convincing evidence exist that they are an established risk factor in OSCC. Therefore more studies are needed in order to clarify the different aspects of virus involvement. Here, we review the existing literature on viral involvement in oral cancer. Ó 2014 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.
Keywords: Oral cancer; Human Papilloma Virus; EpsteineBarr Virus; Human herpes simplex virus type 1
1. Introduction Oral squamous cell carcinoma (OSCC) is the most common epithelial malignancy of the oral cavity. OSCCs and their variants constitute over 90% of oral malignancies, and the disease is associated with poor prognosis. OSCC is a complex malignancy where environmental factors, viral infections, and genetic alterations most likely interact, and thus give rise to the malignant condition. Viral causes of cancer have been studied since the beginning of the 20th century, when an infective agent, which later was shown to be a virus that had the ability to induce tumours in chickens, was isolated [1,2]. During the 1930s, Richard Shope was able to transfer a papillomavirus, by using cell-free medium, to rabbits [3]. Groupe et al. showed in various studies that leukaemia and sarcoma could be induced in mice through an infectious virus [4]. Another major step was made when two research groups successfully transformed cells which were infected with Rous sarcoma virus, into mice [4,5]. It was * Corresponding author. Tel.: þ46 18 6116450. E-mail addresses: [email protected], [email protected] (L. Sand).
thereby possible to identify specific viral oncogenes that could transform normal cells into cancer cells. Viral involvement in the development of OSCC has been proposed in many studies. The most frequently studied virus species are Human Papilloma Virus (HPV), EpsteineBarr Virus and Herpes Simplex Virus Type 1 (HSV-1). Some authors have confirmed a clear connection, while other authors have not been able to find any convincing evidence. Coinfection by two or more virus species has been suggested as an increased risk factor for cancer development in general [6], but also in OSCC [7e10]. There now seems to be a consensus that HPV has oncogenic capacity not only in cervix cancer but also in oral, as well as head and neck cancer. Regarding the other oncogenic viruses, the situation is more doubtful. In this article, we review available literature regarding viral involvement in the development of OSCC. 2. Oral cancer Oral cancer is usually defined as a neoplastic disorder in the oral cavity, which includes the following areas: lip, buccal
http://dx.doi.org/10.1016/j.micinf.2014.02.009 1286-4579/Ó 2014 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved. Please cite this article in press as: Sand L, Jalouli J, Viruses and oral cancer. Is there a link?, Microbes and Infection (2014), http://dx.doi.org/10.1016/ j.micinf.2014.02.009
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mucosa, lower and upper alveolar ridges, retromolar gingiva, floor of the mouth, hard palate, and the anterior two-thirds of the tongue. By gross morphologic examination, one sees exophytic, ulcerative, or verrucous types. Squamous cell carcinoma (SCC) constitute 90% of epithelial malignancy in the oral cavity and over 50% of the tumours have an apparent “precancerous” state, often preceded by potentially malignant disorders such as leukoplakia, oral lichen planus and oral submucous fibrosis [11e13]. The aetiology of epithelial cancer of the head and neck is considered to be a multifactorial, sequential process. The continuum of SCC progress from individual epithelial cell changes (atypia) to a generalized disturbance of the epithelium (dysplasia), and then to carcinoma in situ, and finally to invasive SCC. The malignant tumour, consisting of strands of malignant epithelial cells infiltrating subepithelially, may resemble any or all of the layers of stratified squamous cell epithelium. The two major aetiological factors in SCC of the oral cavity are the social habits of tobacco use and alcohol consumption [14,15]. Different groups of genes are involved in the multiple genetic events of malignant cell transformation: oncogenes, tumour-suppressor genes, DNA repair genes, and DNA sequences that control apoptosis. The normal function of the p53 tumour-suppressor gene, located on the short arm of chromosome 17, is that of “guardian of the genome”. Damage to DNA is associated with nuclear accumulation of the p53 protein, presumably inducing growth arrest for repair or the induction of apoptotic cell death [16]. Mutation in the p53 gene is frequently found in human cancer, and also in SCC of the head and neck. These mutations likely result from carcinogen-induced DNA damage. In patients with head and neck SCC, using high amount of tabacco and alcohol, elevated p53 expression has been detected [17,18]. Elevated p53 has also been associated with HPV-infection [19]. OSCC is treated with surgery, radiotherapy, chemotherapy or a combination of these three modalities. The main prognostic factors are tumour size/stage, presence of locoregional metastasis and sub site and the 5-year survival rate is approximately 50% and has not improved much over the last decades. HPV-infected OSCC, however, seems to have a slightly better prognosis [20]. 3. Oncogenic viruses and oral cancer 3.1. Human Papilloma Virus (HPV) 3.1.1. Historical background Papillomavirus infection in the oral mucosa was first demonstrated in animals by DeMonbreun et al., in 1932 [21]. Yet not until 1967 did Frithiof et al. present the first ultra structural evidence of the presence of papillomavirus in human oral papillomas, and Praetorius-Clausen et al., in 1971, demonstrated particles compatible with papovavirus in oral focal epithelial hyperplasia [22]. Jenson et al. were first to detect HPV antigen in oral verrucae, multiple papillomas, and condylomata [23]. Later, after the development of
hybridization techniques, especially the very sensitive polymerase chain reaction (PCR), numerous investigations have detected different types of HPV in different oral lesions, as well as in normal oral mucosa. 3.1.2. HPV characteristics The papillomaviruses, which replicate in the nucleus of squamous epithelial cells, belong to the papovavirus group, and are small, non-enveloped DNA viruses of a symmetrical icosahedral shape. Papillomavirus particles (52e55 nm in diameter) consist of a single molecule of double-stranded, circular DNA with approximately 8000 bp, contained in a capsid (spherical protein coat) composed of 72 capsomeres (repeating subunits of the capsid) [24]. In a virus, only the genome is present, with no cellular machinery for replicating the genome or manufacturing the capsid. The host cell must supply the necessary ingredients for the assembly of the viral components. When cellular death does not occur during viral reproduction, chronic infection can probably succeed. HPV is a very large and heterogenous group of DNA viruses and over 100 types have already been identified. Based on the clinical behaviour of HPV infections, HPV viruses can be grouped into high-risk (HR) and low-risk (LR) HPV types. HR-HPVs are associated with lesions that have a propensity to undergo carcinogenesis, and these viruses include types 16, 18, 31, 33, 35, 39, 45, and 52. 3.1.3. Carcinogenesis of HPV Initially, HPV infects undifferentiated proliferative basal cells, which are capable of dividing. Once inside the host cell, viral DNA localizes into the nucleus and establishes itself as an episome with a low copy number (some 10e200 copies per cell). At this stage, the viral proteins E1, E2, E6, and E7 transcribed from the early promotor are expressed at a low level [25]. Mainly E6 and E7 may disturb the normal terminal differentiation by stimulating cellular proliferation and DNA synthesis. After the onset of genome amplification, the capsid proteins L1 and L2 accumulate in the mature epithelial cells. The assembly of infectious virions takes place in terminally differentiated cells of the upper epithelial layers, and the virions are shed to the environment, as the cells are lost through desquamation. In addition to active replication, HPV infection can result in latency and malignant transformation through interactions of viral E6 and E7 proteins with p53 and pRB [26]. The tumour suppressor p53 can dictate cellular fate by initiating cell cycle arrest, promoting DNA repair, triggering apoptosis, and inducing growth arrest/senescence. Thus a direct mutation can alter or inactivate p53, and interactions with the oncogene products of HPV (e.g., E6) can also cause aberrations in p53 regulation [27]. The viral E7 protein binds and inactivates a human tumour suppressor gene product, the retinoblastoma protein (pRB) and the viral E6 protein binds p53 and earmarks it for destruction by the ubiquitin pathway. In malignant tumours, including SCC of the head and neck, somatic mutations in the p53 gene, or in other cellular genes that modulate p53 activities, commonly inactivate p53 function. Such E6 elimination of p53 represents a critical step in
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HPV-induced carcinogenesis, in that it provides an alternative way of disrupting the p53 tumour suppressor gene pathway in the cancer cell [28]. The state of the virus (i.e., whether it is maintained as an episome and not integrated, or is integrated into the host genome) seems to play an important role as well. During the normal HPV life cycle, viral DNA is maintained episomally in the nucleus of the affected cell, a state predominantly associated with “low-risk” HPV types such as HPV-6 and -11. 3.1.4. HPV and OSCC The relationship between cervical HPV infection and cervical cancer is well established, and in over 90% of cervical intraepithelial neoplasias (CIN) and cervical cancers, HPV is present [29]. Infection of the cervical epithelium with specific high-risk types of HPV plays a fundamental role in the development of cervical cancer, by causing precursor lesions and maintaining malignant growth [30]. Two review-articles indicated that a similar mechanism is seen in oral potentially malignant disorders [31,32]. Numerous studies have examined the relationship between HPV and SCC in the head and neck region (for review see Hillbertz et al., 2012) [33]. The clear association between HPV and cervical cancer is not as strong for OSCC. HPV integration into host DNA is less common in OSCC [34,35], and in a systematic review, Kreimer et al. found the prevalence of HPV to be 24% in OSCC [36]. Another study reported the estimated proportion of oral and oropharyngeal SCC attributable to HPV infection to be 35% [37]. Na¨sman et al. demonstrated an increasing incidence of HPV-positive tonsillar cancers in the Stockholm area, being 23% in 1970, 68% in 2002, and 93% in 2007 [38]. A strong causal association between HPV infection and tonsil-related cancers was also seen in Montreal [39]. There is also a growing incidence of HPV-associated OSCC in the USA [40], a trend which has been demonstrated in other developed countries [38,41,42]. A prospective study has found that increased risk for head and neck cancer was observed more than 15 years after HPV exposure. Men generally have a higher incidence of oral HPV infection than women but the degree to which tobacco and/or alcohol use increase the risk of HPV-positive OSCC is unclear [43]. However, HPV-positive OSCC may be the main cause of OSCC in non-smokers and non-drinkers. Poor oral health is an additional risk factor for HPV infection, irrespective of smoking and oral sex practice [44]. Further, immunosuppression seems to be an increased risk factor for HPV-positive OSCC [45]. Of the numerous HPV viruses, zur Hausen demonstrated that HPV-16 plays a dominant role in precancerous and cancerous lesions of the cervix, followed by HPV-18 [46]. A similar pattern is seen in OSCC where in most studies, HPV types 6, 11, 16, and 18 have been found, with a clear dominance of HPV type 16 [47e49]. There seem to be enough evidence to claim that HPV is an independent risk factor for OSCC development. It is estimated that 25e35% of all OSCC are caused by HPV.
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3.2. EpsteineBarr Virus 3.2.1. Historical background Dennis Burkitt, a missionary doctor in East Africa in the 1950s, was the first to recognize and describe Burkitt’s lymphoma [50,51]. After a meeting in London in 1961, Dr. Burkitt met Tony Epstein, who together with Yvonne Barr managed to establish Burkitt’s lymphoma-derived cell lines in culture [52]. Examining these cell lines with the electron microscope provided clear evidence of herpes-like particles in a small portion of cells [53]. This virus, distinct from other members of the human herpes virus family, was the first human tumour virus identified. The virus was named the EpsteineBarr Virus. 3.2.2. EBV characteristics The human herpes virus family consists of three subfamilies, i.e., alpha, beta, and gamma. EBV belongs to the gamma subfamily, which is split into two genera, lymphocryptovirus and rhadinovirus. EBV is the prototype for the lymphocryptovirus because it latently infects B-lymphocytes [54]. Like other herpes viruses, EBV has a toroid-shaped protein core, wrapped with DNA; a nucleocapsid with 162 capsomeres; a protein tegument between the nucleocapsid and the envelope; and an outer envelope with external glycoprotein spikes. The EBV stores its genome in the form of a linear, double-stranded, 172 kbp DNA molecule [55]. Two EBV subtypes have been identified, i.e., EBV-1 and EBV-2, differing in the genes coding for nuclear proteins EBNA-LP, 2, -3B, and -3C, with differences in the predicted amino acid sequence of between 28% and 47% [56]. 3.2.3. Carcinogenesis of EBV EBV is one of the most common and widespread human viruses, infecting over 95% of people worldwide. After acute infection with or without symptoms, latent EBV infection will persist lifelong. Hence, EBV has the ability to establish a latent infection, which means a silent state of viral infection characterized by the low expression of viral genes and minimal cytopathic effects or production of infectious virus. EpsteineBarr Virus has been identified as the causative agent of infectious mononucleosis. EBV enters the host through the oropharynx and has developed a way to simultaneously access both B cells and squamous epithelial cells of the oropharynx. However, B cells are considered necessary and sufficient for EBV infection, while epithelial cells may be seen as helpful enhancers for transferring the virus to others, and for establishing latency in B cells [57]. In most cases, transmission occurs orally via the saliva. EBV gene products synthesized in latent infections are of particular interest in relation to the oncogenic properties of this virus. A number of gene products have been characterized that are expressed in specific tumours or in non-transformed, latently infected cells. Some of these share structural and functional homologies with cellular gene products. The nuclear EBNA-1 protein is consistently expressed; this is a sequence-specific DNA-binding phosphoprotein that plays a central role in the episomal maintenance of the EBV genome and is required for its DNA replication [58].
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EBV is associated with “oral hairy leukoplakia”, which is a non-malignant white lesion at the lateral boarder of the tongue in immunodeficient individuals [59]. Burkitt’s lymphoma (BL), a non-Hodgkin, monoclonal Blymphocyte tumour of low differentiation grade, frequently presents in the jaw, and is found endemically in equatorial Africa and Papua New Guinea. Almost 50% of the affected patients are children. More than 90% of the BL in these areas contain EBV genomes. Outside these areas, only sporadic cases are seen. In sporadic BL, EBV can be detected in only 10e20% of the tumours [60]. Nasopharyngeal carcinoma (NPC), an epithelial cell tumour that develops in the space behind the nose that is rich in lymphocytes is endemic in people from Southern China; native North Americans; Tunisians; and East Africans. In Cantonese Chinese and native North Americans, 20% of all cancers may be NPC, whereas in Western countries, less than 1% of all cancers are NPC [60]. In contrast to BL, EBV is present in every anaplastic NPC cell. Genetic predisposition, environmental factors, and nutritional factors are thought to be important in NPC aetiology [61]. 3.2.4. EBV and OSCC In vivo, EBV infects two types of target cells in the oral region: epithelial cells in the oro/nasopharynx and/or salivary gland, and the B-lymphocytes [62]. Thus, this has been the reason for the suspicion that EBV is involved in OSCC and premalignant lesions. Using PCR, variability in EBV prevalence has been high in previous studies of OSCC, premalignant oral lesions, and normal oral mucosa [63,64]. EBV has been associated with OSCC in some studies [8,65,66], while in other studies no clear association between EBV and OSCC has been found [67,68]. In 145 OSCC samples from toombak users in Sudan, EBV was detected in 53 (37%) of the samples. The corresponding figure for the OSCC samples from nontoombak users was 16 (22%) for EBV [10]. EBV DNA was detected in 18 (29%), from 62 Indian patients with betelassociated OSCC [9]. This association between smokeless tobacco and EBV was not found in a study from Sweden on patients with snuff-induced lesions [64], and even though Sand et al. found a significant increase in EBV prevalence in OSCC compared to healthy controls, no correlation with tobacco use was seen [65]. While some authors found a clear difference in EBV positivity between oral lesions and controls, others found higher EBV prevalence in the controls. This high variability and inconsistency probably reflects differences in ethnicity, geographical differences as well as differences in methodological sensitivity [9,10]. The variable EBV prevalence in OSCC in different studies might be explained by different sampling techniques, divergent PCR methods and also the quality of the sample (e.g. whether frozen or fixed). The prevalence of EBV in oral samples varies widely between ethnic and geographical populations. In a study by Jalouli et al., 85 (55%) of 155 examined OSCCs from 8 different countries were EBV-positive, but the variation between countries was large (22e80%) [63]. Southeast Asian studies
found a high prevalence of EBV and suggested an aetiological role of EBV in OSCC [69]. Through immunohistochemical detection of LMP-1, the EBV antigen was associated with transforming cell activity in an Egyptian population [70]. By contrast, North American studies, as well as West and North European studies, regularly report lower prevalence of EBV [67]. Even though there are plenty of reports showing an association between EBV and OSCC development, no safe conclusions could be drawn from those. Further, many reports show no association. More and larger population based studies are needed to prove the oncogenic potential of EBV in OSCC. 3.3. Herpes Simplex Virus Type 1 (HSV-1) 3.3.1. Historical background The clinical manifestation of oral herpetic infections has been known for 250 years, but the causative agent was not identifies as a herpes virus until the 20th century. In 1962, Schneweis identified two serotypes [71], subsequently confirmed by Nahmias and Dowdle [72]. A casual association between a herpes virus and renal adenocarcinoma of the leopard frog was suggested by Lucke´ in 1938 [73]. Additional experimental studies seemed to support a possible role of HSV in human cancers. These originated in part from reports of HSV-DNA or RNA persistence in cervical cancer cells: in 1972, Frenkel et al. and in 1973, Roizman and Frenkel, identified a fragment of HSV-2 in one cervical cancer biopsy and also analysed transcripts of this DNA [74,75]. 3.3.2. HSV-1 characteristics Herpes simplex virus belongs to a family of eight related viruses, including herpes simplex virus types 1 (HSV-1) and 2 (HSV-2), varicella-zoster virus, EpsteineBarr Virus, and cytomegalovirus. Like all herpes viruses, HSV-1 and the closely related HSV-2 have enveloped, spherical virions. The genome of HSV-1 is densely packaged in a liquid-crystalline, phage-like manner in a 100-nm icosahedral capsid [76]. The genome of HSV-1 consists of linear double-stranded DNA, with approximately 150 000 bp. A significant property of all herpes simplex viruses is their ability to remain latent in host neurons for life, and can reactivate to cause lesions at or near the site of initial infection. 3.3.3. Carcinogenesis of HSV-1 Infection with herpes simplex viruses (HSV) is very common worldwide. In European countries, the estimated prevalence of HSV-1, determined by means of antibody measurements, varies between 60% and 90% in the general population [77]. The prevalence of HSV-1 exceeds 40% by age 15 years and rises to 60e90% in older adults. In the USA, there are estimated to be approximately 500 000 primary infections per annum [78]. After inoculation of the skin or mucous membrane, HSV-1 is transported along sensory axons in a retrograde direction to the neuronal cell body, where it establishes lifelong latent infection. Periodic reactivation results in HSV-1 being transported in an anterograde direction to
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nerve terminals, where it causes recurrent clinical disease or asymptomatic viral shedding [79]. HSV-1 infection is primarily oro-pharyngeal and HSV-2 infection is primarily genital, even though HSV-1 has been found in genital lesions and HSV-2 in oral lesions. Facial herpes may establish latency in the trigeminal ganglion, while genital herpes may involve the sacral ganglion. Herpes simplex infection generally occurs in two phases: the initial, primary infection, followed by secondary, recurrent disease at the same site. In the first phase, the virus spreads by close person-to-person contact with lesions or mucosal secretions (e.g., saliva or cervical discharge) as well as via respiratory droplets. The incubation period is 2e10 days; the virus then spreads to regional lymph nodes, causing tender lymphadenopathy [80]. Though HSV-1 infections are frequently asymptomatic, they can produce a variety of signs and symptoms. These include oral or perioral lesions, ocular infections, congenital skin lesions, genital skin or mucous membrane lesions, and serious systemic illnesses such as encephalitis and neonatal disease. One of the major features of HSV infection is the ability of the virus to remain latent in the sensory ganglia. Reactivation from the latent state can be triggered by external stimulus and results in axonal transportation of the virus back to the epithelium, leading to either clinical lesions with lytic (productive) destruction of distal epithelial cells or subclinical shedding. Increased antibody levels against HSV-2 have been reported on women with cervical cancer. A large-scale seroepidemiological study by Smith et al. linked HSV-2 seropositivity with increased risk of cervical squamous cell carcinoma [81]. On the other hand, a Scandinavian study on 550 000 women come to the opposite conclusion [82]. Integration of HSV DNA in tumours would be an indication of HSV involvement in carcinogenesis. There have been reports on HSV DNA and RNA in cervical tumours, while several studies have not been able to detect any HSV DNA or RNA in malignant cervical lesions. However, several studies have shown regions of homology between HSV and human DNA, which make the interpretation of these studies difficult. A “hitand-run” mechanism has also been proposed for HSV oncogenesis [83]. 3.3.4. HSV-1 and OSCC The role of HSV in the development of OSCC has been investigated in both animal models and in vivo. In the first animal model on this topic, Southam et al. demonstrated that HSV could be a co-carcinogen. They showed that an infection with HSV-1 could increase the proportion of animals with carcinomas from 7 to 19% [84]. Subsequently, Duff and Rapp showed that if the virus was partially inactivated with various agents to prevent it from killing cells, a certain number of the surviving cells would then become transformed to a malignant phenotype. These transformed cells did not only invade local tissue but also metastasize to distant sites in the animals [85]. In a study of hamsters, Stich et al. demonstrated that snuff significantly enhanced herpes simplex virus-associated development of micro-invasive SCC in cheek pouch epithelium
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[86]. In a study of rats, Larsson et al. demonstrated that HSV-1 and snuff interact in the development of malignant lesions [87], and Hirsch et al. demonstrated that the incidence of malignant tumours was significantly higher in rats exposed to snuff and HSV/snuff than in control animals. Further, exposure to both the tobacco and the virus produced more tumours than exposure to either agent alone. One explanation would be that HSV-1 potentiates tumours that are induced by carcinogens in the tobacco [88]. Cornella et al. used an animal model where hamsters were exposed to intraoral infections with HSV-1, and after recovery, the cheek pouches were exposed to the carcinogen 7,12dimethyl-benz[a]anthracene [DMBA]. The number of tumours was significantly higher in animals that were previously infection with HSV-1 [89]. A similar situation to humans would be where HSV-1 becomes latent in the trigeminal ganglion after which the oral mucosa is exposed to tobacco carcinogens. Clinical studies have shown a possible interaction between smokeless tobacco and HSV-1 in the development of OSCC [7]. Jalouli et al. investigated the presence of HSV-1 DNA in tobacco-related OSCC from a Sudanese and an Indian population [9,10]. In toombak-related OSCCs, HSV-1 was detected in 29% of the samples, compared to 24% in nontoombakrelated OSCCs and HSV-1 DNA was detected in 5% of the 62 patients with betel-related OSCC. In a subsequent study they also compared OSCCs from 8 different countries regarding HSV-1 [63]. The overall prevalence of HSV-1 positive OSCC was 15%. However, the difference between the countries was large, with the UK having the highest prevalence (55%). There was a statistically significant higher HSV-1 prevalence in the industrialized countries compared to the developing countries, mainly due to the high prevalence in the UK. No safe conclusions could be drawn from these studies but the ethnic composition and socio-economic situation seem to be important factors regarding the prevalence of HSV-1 in OSCC. Meurman claim in a review study from 2010, that HSV infections link statistically with oral carcinogenesis and that antibody levels to HSV-1 and HSV-2 are increased in OSCC patients [90]. A population-based study from USA, suggests that HSV-1 may enhance the development of OSCC [91]. However, another American study came to the opposite conclusion where the risk of OSCC was not significantly increased in those with HSV-1 or HSV-2 antibodies compared to HSV-negative patients [92]. Taken together, no evidence that HSV acts as a direct carcinogen in OSCC exists to date. The transformation studies were mostly performed 2e3 decades ago, and the results of recent studies are inconclusive and contradictory. 4. Viral detection methods Several methods for detection of viral DNA have been used, including PCR assays, in situ hybridization (ISH), immunohistochemical staining (IHC), dot blotting and southern blotting [93].
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Before the era of molecular techniques, serological tests were common for detection of EBV and HSV-1. For detection of HPV infection, serology is an unreliable method, because frequent cross-reactions and transient antibody responses reduce sensitivity and specificity, which therefore is low, especially when compared to PCR. Increased antibody titres against EBV in serological tests are strongly correlated with EBV induced malignancies such as NPC. EBV associated tumours are often characterized by early antigens and IgG viral capsid antigens with diminished EBNA titres. However, this pattern is not specific for malignancy, implying that serology alone is inadequate for diagnosing EBV-related malignancies. The main advantages of molecular techniques are its higher sensitivity and specificity. In situ hybridization (ISH), widely used for virus detection, can be utilized on formalin-fixed and paraffin-embedded sections from biopsy specimens. ISH has the advantage of providing information on whether HPV is present, and if so, where HPV DNA is located in the tissue. In addition, information about morphological abnormalities associated with HPV can then be provided. ISH, which can localize latent EBV in tissue samples, has been used for detection of EpsteineBarr encoded RNAs in investigations of the association between EBV and malignancies [94]. ISH has a disadvantage of low sensitivity, compared to PCR, with a detection limit of about 20 viruses per infected cell, and ISH is also more time consuming. Southern blot hybridization (SBH) is a process in which complementary single strands of nucleic acids combine to form a stable double-stranded nucleic acid molecule. The main disadvantages of SBH compared with PCR are low sensitivity, large amounts of cellular DNA required, and greater expense. However, despite these drawbacks, SBH is useful for confirming PCR results, especially in the case of paraffin-embedded tissue samples, in which background amplification bands may preclude interpretation of data from ethidium bromide staining. The polymerase chain reaction (PCR) assay is a technique that offers several advantages over other methods. It requires only a small quantity of biological material and can detect the viral presence in “early” infections. PCR detection of HPV, EBV, and HSV is highly sensitive and specific, and can supplement the detection of clinical manifestations of virusassociated oral lesions [95]. PCR and variants of PCR are currently the most commonly used viral detection method. The disadvantage however, is the risk of contamination and internal controls are of most importance in PCR-based assays. Additionally, several methods and newer techniques are constantly under investigation. 5. Conclusions There now seems to be a consensus that HPV has oncogenic capacity not only in cervical cancer but also in oral, as well as head and neck cancer. Review-studies and metaanalyses indicate that approximately 25e30% of oral cancer could be attributed to HPV and HPV type 16 is the most important subtype in oral carcinogenesis. Regarding the other
oncogenic viruses, the situation is more doubtful. At present, it would be fair to claim that the aetiological role of EBV in oral SCC is highly questionable. If HSV plays an active role in OSCC development or if it is only a passive by-stander in the decreased local immune-deficient tumour area, remains unclear. The fact that some studies indicate that co-infection by multiple oncogenic viruses may be important risk factors is OSCC development needs further investigation. PCRbased assays are currently the mostly used viral detection method. References [1] Ellerman C, Bang O. Experimentelle Leuka¨mie bei Hu¨hnern. Cent Bakteriol 1908;46:595e609. [2] Rous P. A sarcoma of the fowl transmissible by an agent separable from the tumor cells. J Exp Med 1911;13:397e411. [3] Shope R. Serial transmission of virus of infectious papillomatosis in domestic rabbits. Proc Soc Exp Biol Med 1932;32:830e2. [4] Groupe V, Manaker R. Discrete foci of altered chicken embryo cells associated with Rous sarcoma virus in tissue culture. Virology 1956;2:838e40. [5] Temin H, Rubin H. Characteristics of an assay for Rous sarcoma virus and Rous sarcoma cells in tissue culture. Virology 1958;6:669e88. [6] Yang YY, Koh LW, Tsai JH, Tsai CH, Wong EF, Lin SJ, et al. Correlation of viral factors with cervical cancer in Taiwan. J Microbiol Immunol Infect 2004;37:282e7. [7] Yang YY, Koh LW, Tsai JH, Tsai CH, Wong EF, Lin SJ, et al. Involvement of viral and chemical factors with oral cancer in Taiwan. Jpn J Clin Oncol 2004;34:176e83. [8] Al Moustafa AE, Chen D, Ghabreau L, Akil N. Association between human papillomavirus and Epstein-Barr virus infections in human oral carcinogenesis. Med Hypotheses 2009;73:184e6. [9] Jalouli J, Ibrahim SO, Mehrotra R, Jalouli MM, Sapkota D, Larsson PA, et al. Prevalence of viral (HPV, EBV, HSV) infections in oral submucous fibrosis and oral cancer from India. Acta Otolaryngol 2010;130:1306e11. [10] Jalouli J, Ibrahim SO, Sapkota D, Jalouli MM, Vasstrand EN, Hirsch JM, et al. Presence of human papilloma virus, herpes simplex virus and Epstein-Barr virus DNA in oral biopsies from Sudanese patients with regard to toombak use. J Oral Pathol Med 2010;39:599e604. [11] Schepman K, der Meij E, Smeele L, der Waal I. Concomitant leukoplakia in patients with oral squamous cell carcinoma. Oral Dis 1999;5:206e9. [12] Noonan VL, Kabani S. Diagnosis and management of suspicious lesions of the oral cavity. Otolaryngol Clin North Am 2005;38:21e35. [13] Gupta PC, Bhonsle RB, Murti PR, Daftary DK, Mehta FS, Pindborg JJ. An epidemiologic assessment of cancer risk in oral precancerous lesions in India with special reference to nodular leukoplakia. Cancer 1989;63:2247e52. [14] Proia NK, Paszkiewicz GM, Nasca MA, Franke GE, Pauly JL. Smoking and smokeless tobacco-associated human buccal cell mutations and their association with oral cancer e a review. Cancer Epidemiol Biomarkers Prev 2006;15:1061e77. [15] Hirsch JM, Wallstrom M, Carlsson AP, Sand L. Oral cancer in Swedish snuff dippers. Anticancer Res 2012;32:3327e30. [16] Koh J, Cho N, Kong G, Lee J, Yoon K. p53 mutations and human papillomavirus DNA in oral squamous cell carcinoma: correlation with apoptosis. Br J Cancer 1998;78:354e9. [17] Ibrahim S, Johannessen A, Idris A, Hirsch J, Vasstrand E, Magnusson B, et al. Immunohistochemical detection of p53 in non-malignant and malignant oral lesions associated with snuff dipping in the Sudan and Sweden. Int J Cancer 1996;68:749e53. [18] Robinson M, Sloan P, Shaw R. Refining the diagnosis of oropharyngeal squamous cell carcinoma using human papillomavirus testing. Oral Oncol 2010;46:492e6. [19] Dai M, Clifford GM, le Calvez F, Castellsague X, Snijders PJ, Pawlita M, et al. Human papillomavirus type 16 and TP53 mutation in oral cancer:
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