CME
Amanda Psyrri, MD Daniel DiMaio, MD
Nat Clin Pract Oncol 5(1):2431, 2008. © 2008 Nature Publishing Group
Cervical cancer is a major cause of cancer mortality in women worldwide and is initiated by infection with high-risk human papillomaviruses (HPVs). High-risk
HPVs, especially HPV-16, are associated with other anogenital cancers and a subgroup of head-and-neck cancers. Indeed, HPV infection could account for the development of head-and-neck cancer in certain individuals that lack the classical risk factors for this disease (tobacco and alcohol abuse). This Review summarizes the main events of the HPV life cycle, the functions of the viral proteins, and the implications of HPV infection on their hosts, with an emphasis on carcinogenic mechanisms and disease outcomes in head-and-neck cancer. The demonstration that HPVs have a role in human carcinogenesis has allowed the development of preventive and therapeutic strategies aimed at reducing the incidence and mortality of HPV-associated cancers.
Papillomaviruses are small non-enveloped DNA viruses that infect squamous epithelial cells. More than 200 papillomavirus types have been isolated and there are certainly additional types that have not yet been identified. These viruses have been found in many organisms, including humans. Human papillomaviruses (HPVs) give rise to a large spectrum of epithelial lesions, mainly benign hyperplasia (e.g. warts or papillomas) with low malignant potential.
There is a subgroup of HPVs, the ‘high-risk’ HPVs, which are associated with precancerous lesions. A small fraction of people infected with high-risk HPVs will develop cancers, which usually arise many years after the initial infection.
On the basis of epidemiological and molecular evidence, in 1995, the International Agency for Research on Cancer recognized that the highrisk HPV types 16 and 18 were carcinogenic in humans.
[1] Together, these two HPV types are responsible for approximately 70% of cervical cancer cases, and other high-risk
HPV types account for virtually all of the remaining cases of this disease.
[2] Cancer of the uterine cervix is, therefore, the malignancy most widely accepted as being associated with HPV infection. In addition, high-risk HPVs are associated with other anogenital carcinomas, including vulvar, anal, and penile cancers [3,4] and, as described in detail herein, some head-and-neck squamous cell carcinomas (HNSCCs).
[5]
High-risk HPVs cause cervical dysplasia and carcinoma in situ , which if locally confined can be successfully treated but if left untreated can progress to cervical carcinoma. There is usually a latency period of 10 years or more between the initial high-risk HPV infection and the development of cervical cancer.
[6]
This prolonged natural history allows preinvasive cervical lesions to be diagnosed using Pap smear screening programs. These screening programs have significantly decreased the incidence of cervical cancer in the developed world, but have not been widely adopted in the developing world. This inability to establish effective Pap smear programs in certain parts of the world contributes to the high incidence of cervical disease in these areas. Presumably, the combination of Pap smear screening with HPV-DNA testing will improve our ability to stage and manage cervical lesions. In addition, prevention of high-risk
HPV infection by prophylactic vaccination is likely to prevent many HPV-associated cancers.
HPV-associated cancers maintain and express the HPV viral genes even in advanced stages of disease, and repression of viral oncogene expression can prevent the growth or survival of cervical cancer cells. This finding raises the possibility that even late-stage cancers can be cured by HPV-targeted strategies, such as medicines that interfere with the expression or action of viral proteins and therapeutic vaccines that elicit a cytolytic immune response to cells expressing these proteins. This Review describes the principles of HPV-induced carcinogenesis in relation to cervical and head-and-neck carcinomas.
The HPV genome is a double-stranded circular DNA molecule of 8,000 base pairs (bp) that encodes up to ten proteins. Only one DNA strand is transcribed into mRNA. The genome is divided into three portions: a
∼
4,000 bp region that encodes proteins primarily involved in viral DNA replication and cell transformation; a ∼ 3,000 bp region that encodes the structural proteins of the virus particles; and a ∼ 1,000 bp noncoding region that contains the origin of viral
DNA replication and transcriptional regulatory elements.
[7]
Through wounds or abrasions, the papillomaviruses infect basal epithelial cells, which are the only actively dividing cells in the epithelial layer. The viral DNA is maintained in the nuclei of infected basal epithelial cells as a low-copy-number plasmid.
[8] Squamous epithelial cells normally undergo differentiation as they move from the basement membrane towards the surface epithelium, and HPV-DNA replicates to a high copy number only in terminally differentiated cells near the epithelial surface.
[8,9] Similarly, the late viral genes, which encode the L1 and L2 proteins that constitute the virus particle, are expressed only in the highly differentiated cells, where infectious progeny virus is produced and released.
[8]
Replication of the HPV genome is critically dependent on the host-cell DNA replication machinery, as is seen with other small DNA viruses. Although terminally differentiated epithelial cells are normally not able to support DNA synthesis, viral DNA replication takes place in these cells — HPVs encode E6 and E7 proteins that create a state competent for DNA replication.
[10] The E6 protein of the high-risk HPV binds and induces the degradation of the p53 tumor suppressor protein via a ubiquitin-mediated process, [11,12] while the HPV-E7 protein binds and destabilizes the retinoblastoma (Rb) tumor suppressor protein and related proteins.
[13,14] The E6 and E7 proteins also interact with other cellular targets.
[7] Together, these effects promote cell-cycle progression and viral
DNA replication in differentiated keratinocytes.
[15,16]
The papillomavirus E1 and E2 proteins are required for viral DNA replication and papilloma formation.
[17] E1 is an ATP-dependent helicase that initiates viral replication in cooperation with the E2 protein.
[18-20] In addition, the E2 protein can function as a transcriptional repressor of E6 and E7 oncogene expression, [21] and has an essential role in the segregation of viral DNA plasmids as cells divide through its association with cellular chromatin-associated proteins.
[22,23]

The observation in 1842, in Italy, that cervical cancer developed almost exclusively in married women and was rare in nuns, was the first hint that a sexually transmitted agent was associated with the pathogenesis of cervical cancer.
[24]
Harald zur Hausen’s laboratory was the first to demonstrate that cervical cancer biopsies and cervical cancer cell lines contained HPV-DNA sequences.
[25-27] HPV-16 is the most prevalent high-risk HPV type and is found in approximately
50% of cervical cancer cases. HPV-18 is the second most prevalent high-risk HPV, with other HPV types being isolated from fewer cases of cervical cancer.
[2]
Several lines of evidence indicate that high-risk HPV types have an essential role in cervical carcinogenesis. First, almost all cervical carcinomas contain and express high-risk HPV sequences, frequently in an integrated form.
[2,28,29] Second, epidemiological studies demonstrated that persistent infection with a highrisk HPV dramatically increases the risk of cervical carcinoma.
[28,30] Third, molecular and cellular studies demonstrated that high-risk HPVs encode the E6 and
E7 oncoproteins, which interfere with well-established tumor suppressor pathways.
[7,31] Fourth, the E6 and E7 oncogenes are continuously expressed in cancer cells and are required for proliferation and survival of cervical cancer cell lines.
[7,32-34] Fifth, transgenic mice expressing high-risk HPV sequences develop cervical carcinoma following exposure to estrogen.
[35,36] Finally, vaccination that prevents persistent high-risk HPV-16 and HPV-18 infection in women also prevents the development of precancerous cervical lesions.
[37-39]
The continuous expression of high-risk E6 and/or E7 genes causes genomic instability, due in part to the ability of the viral proteins to disrupt tumor suppressor pathways.
[7,40] In addition, the E6 oncoprotein interferes with DNA repair enzymes, and the E7 oncoprotein can induce structural and numerical chromosome abnormalities by the disruption of centrosome synthesis.
[41,42] This genetic instability can cause the emergence of tumorigenic cells. Despite the accumulation of cellular mutations during carcinogenesis, repression of E6 and E7 expression in cervical carcinoma cell lines results in restoration of Rb and p53 activity and is sufficient to induce cell growth arrest or apoptosis.
[32,33,43]
In addition to the strong association between HPV infection and cervical carcinoma, mounting epidemiological, molecular and clinical evidence indicates that high-risk HPVs (especially HPV.16) account for the development of headand-neck carcinoma in some individuals who do not have the classical risk factors for this disease (i.e. a history of tobacco use and/or alcohol consumption).
[5,44.47] According to Surveillance, Epidemiology, and End Results (SEER) data, the annual incidence of base-of-tongue and tonsil cancers in the USA increased by 2.1% and 3.9%, respectively, from 1973 to 2001 among white men and women aged 20-44 years, whereas incidences at other sites declined.
[48,49] Similarly, the incidence of tonsillar cancer increased by approximately 2-3% per year among men younger than 60 years from 1975 through 1998.
[50] This increase in the incidence of oropharyngeal cancer was paralleled by an increase in certain sexual behaviors. In the US, between the periods 1976-1980 and 1988-1994, herpes simplex-2 seroprevalence, a surrogate marker of sexual activity, increased by 30%.
[51] This change in the demographics of patients with head-and-neck cancer is consistent with a role for genital HPVs in the pathogenesis of oropharyngeal squamous cell carcinoma (OSCC) in individuals whose sexual practices are typically associated with sexual transmission of the virus. Several studies suggest that oral HPV infection is sexually acquired.
[44,52] A recent hospital-based case-control study of 100 newly diagnosed patients with OSCC and
200 control patients without cancer revealed that a high (i.e. 26 or more) lifetime number of vaginal-sex partners and 6 or more lifetime oral-sex partners were associated with OSCC (odds ratios 3.1 and 3.4, respectively).
[44] The authors conclude that there is a strong association between oral HPV infection and
OSCC among subjects with or without tobacco or alcohol use, the established risk factors for this disease. Although oral —genital contact may be responsible for HPV transmission, transmission through direct mouth-to-mouth contact or other means could not be excluded. An increased risk of HPV-associated OSCC in individuals with a history of HPV-associated anogenital cancers and in husbands of women with in situ carcinoma and invasive cervical cancer also suggests that sexual transmission of HPV infection to the oral cavity can occur.
[47,53]
Markers of HPV infection are also associated with increased risk of OSCC.
[44,54,55]
D’Souza et al . showed that OSCC was significantly associated with oral HPV infection and with HPV-16 L1 seropositivity among patients both with and without a history of heavy tobacco and alcohol use.
[44] In a Swedish study, oral highrisk HPV infection was associated with dramatically increased risk of OSCC development (odds ratio 230; 95% CI 44-1,200), after adjustment for alcohol and tobacco use.
[55] In a separate, nested case-control study, HPV-16-seropositivity conferred a greater than 14-fold increased risk of subsequent development of
OSCC.
[54] Studies that failed to show an association between sexual habits, oral HPV infection and HNSCC include cohorts with a less than 25% incidence of
HPVDNA positivity, where the risk association might be attenuated.
[52,56] Studies that restricted enrollment to patients with OSCC showed a clear association between sexual behaviors, oral HPV infection and HNSCC.
[44,46,57] Tonsillar crypts seem particularly susceptible to transformation by HPV, which is similar to the transformation zone of the uterine cervix, the location in which most cervical cancers originate.
[58]
Establishing the link between HPV and a subset of OSCC has been difficult because of the heterogeneity of OSCC, and the fact that only a fraction of cases are HPVassociated. Syrjänen et al . observed that some oral squamous cell cancers have morphological and immunohistochemical features indicative of
HPV infection.
[59] This finding was the first hint that HPV may be involved in the pathogenesis of a subset of HNSCC.
[59] Since HPV-16 DNA was first detected in an invasive HNSCC in 1985, [60] HPV sequences have been repeatedly detected in a variable proportion of HNSCC, from as few as 10% to 100%.
[61] This disparity might reflect the different anatomic locations of tumors and the techniques used to detect HPV-DNA. HPV-associated OSCC tend to be poorly differentiated, often basaloid in histology, and frequently present at an advanced stage.
HPV-16 is the most prevalent genotype in cervical carcinoma, and is also the most frequently detected HPV type in HNSCC, found in up to 90% of HPVpositive cases.
[5,45] Some reports have indicated that the HPV subtypes associated with OSCC are similar but not identical to HPVs found in cervical carcinoma.62-64 Chen et al . showed that HPV-associated HNSCC harbor HPV-16 subtypes with characteristic changes in their promoter/enhancer region that render them particularly active in oral keratinocytes.
[62]
Transcription of HPV-16 E6/E7 mRNA in tonsillar carcinomas is not necessarily dependent on viral DNA integration, and the viral DNA is predominately in episomal form.
[65] It is not clear how the viral DNA can remain in cancer tissues as episomes with high copy numbers. A report by Van Tine and coauthors provided evidence that the HPVE2 protein might serve as an ‘anchor’ to bind episomal HPV to cellular mitotic spindles, thus, ensuring maintenance of the episomal state.
[66]
In a mechanism similar to that seen in cervical cancer, viral oncoprotein-mediated abrogation of the p53 and pRb pathways obviates the need for mutational inactivation of TP53 and RB1 genes in HPV-associated OSCC. HPV-induced cancers are, therefore, associated with wild-type TP53 and RB1 genes and low levels of p53 and pRb proteins. The p16 tumor suppressor gene (CDKN2A) is also often upregulated in these cancers because it is negatively regulated by pRb.
[67,68] Overexpression of p16 has been repeatedly reported in HPV-associated cancers. In one study of cervical and genital lesions, high levels of p16 protein expression were associated with high-risk HPV infection.
[69] A recent study of lymph-node metastases in HNSCC reported that p16 protein overexpression is a surrogate marker for oropharyngeal origin and HPV-association.
[70] By contrast, loss of p16 protein expression is a common and early event in tobacco-related HNSCC. Thus, tobacco/alcohol-associated OSCC are associated with downregulation of p16 protein and TP53 gene mutation, [71] whereas HPV-associated OSCC are associated with wild-type TP53 and RB1 genes and upregulation of p16 protein levels.

Several lines of clinical evidence also suggest that HPV-associated OSCC could be biologically distinct from classical OSCC.
[45,72] Tobacco-associated OSCC are more frequent in men, while men and women are at equal risk of HPV-associated OSCC. In addition, patients with HPV-associated OSCC are often nonsmokers and nondrinkers and on average 5 years younger than their tobacco-use-associated counterparts.
[58]
HPV-DNA detection per se in an OSCC does not prove causal association. Only HPV DNA that is transcriptionally active is biologically and clinically relevant.
To address the role of HPV-16 expression in OSCC, we studied a cohort of 107 OSCC samples for HPV-16-DNA viral load using real-time PCR.
[45] In addition, we constructed a tissue array composed of these tumors and studied expression of p53, pRb and p16 using a quantitative in situ method of protein analysis. We hypothesized that for HPV-DNA-positive cases, p16 expression status would identify those that were biologically relevant. These experiments delineated three biologically and clinically distinct types of OSCC: class I, HPV-negative/p16 nonexpressing; class II, HPV-positive/p16 nonexpressing; and class III, HPV-positive/p16 expressing oropharyngeal tumors. Only patients in class III had significantly lower p53 and pRb expression. The 5-year survival in class III was 79%, significantly higher than in the other two classes (20% and 18%, P = 0.0095). Diseasefree survival for class III was 75% compared with
15% and 13% for classes I and II, respectively (P = 0.0025). The 5-year local recurrence was 14% in class III compared with 45% and 74% (P = 0.03).
Multivariate survival analysis confirmed the prognostic value of the threeclass model. Thus, only the HPV-positive/p16 expressing OSCC tumors (class III) fit the cervical carcinogenesis model, and these tumors are associated with a favorable prognosis.
The classic oncogenic insult in HNSCC has been attributed to excess alcohol or tobacco exposure producing mutational or epigenetic inactivation of TP53, p16 and RB1 in a multistep progression from normal cell to dysplasia to carcinoma. On the basis of our results, we can refine a model for HPV-associated oropharyngeal cancer.
[72] In these HPV-induced tumors, oncogenic HPV-E6 and HPV-E7 proteins inactivate p53 and pRb pathways, with subsequent upregulation of p16 expression. This model eliminates the need for mutational inactivation of the RB1 and TP53 genes and the pathways they control. In addition to the previously described models, [72] we also describe a novel class II of OSCC, where HPV-16 DNA is present, but p53, p16 and pRb expression is similar to class I tumors that lack HPV-DNA.
[45] These class II tumors might arise when tobacco/alcohol-related tumors are infected by high-risk HPVs. It is not clear whether these tumors represent a group that is biologically distinct from HPV-negative tumors.
HPV-associated OSCC are associated with a better prognosis than HPV-negative tumors in the majority of studies.
[5,73.78] In our study, HPVpositivity confers a
60% to 80% reduction in risk of death from cancer compared with HPVnegative tumors.
[45] What are the clinical implications of a role of sexually transmitted
HPV in a subset of head-and-neck cancers with a relatively favorable prognosis? Prophylactic vaccines that prevent persistent cervical HPV.16 infections might be effective in preventing these cases of head-and-neck cancer as well, either indirectly by eliminating an anogenital source of virus or directly by protecting the oropharyngeal epithelium itself from infection. Similar benefits could arise from strategies that inhibit viral replication; for example, from drugs that block the ability of the E1 and E2 proteins to drive viral DNA replication. In addition, viral etiology might provide opportunities to treat established cancers.
Thus, antiviral measures being developed to treat cervical cancer may well be effective in HPV-associated OSCC.
Detection of high-risk E6/E7 mRNA or protein would be the ideal test for classifying a tumor as truly HPV-associated, but this determination is not feasible in formalin-fixed, paraffin-embedded tissue. Determination of p16 expression status by immunohistochemistry could serve as a reasonable surrogate marker for biologically relevant high-risk HPV infection. The addition of p16 immunohistochemistry to existing protocols to determine HPV DNA presence may, therefore, prove useful in the classification of OSCC.
The favorable outcome of HPV-induced oropharyngeal cancers might be attributable to the absence of field cancerization or enhanced radiation sensitivity.
[77]
The term ‘field cancerization’ is used to describe the presence of carcinogeninduced early genetic changes in the epithelium from which multiple independent lesions arise, leading to the development of multifocal tumors.
[79] In addition, unlike tobacco-associated oropharyngeal cancers that harbor mutant TP53, the apoptotic response of HPV-associated tumors to radiation and chemotherapy might be intact, as seems to be the case in some cervical cancers.
[80] It might be important to distinguish HPV-associated HNSCC with favorable prognosis from poor prognosis tobacco/alcohol-associated HNSCC, especially as specific anti-HPV treatment becomes available. Careful clinical trials are required to determine the optimum management of HPV-associated HNSCC, compared with
HPV-independent HNSCC. The diagnosis of HPV-associated OSCC should be considered in all OSCCs, especially those arising from the tonsils in patients without a history of tobacco use or alcohol abuse, and in immunocompromised patients and those with basaloid or poorly differentiated tumors.
As described in detail in a recent review, prophylactic HPV vaccines have been developed on the basis of recombinant expression and selfassembly of the major capsid protein, L1, into immunogenic virus-like particles that resemble authentic virions but are noninfectious.
[81] One of the vaccines, Gardasil® (Merck
& Co., Whitehouse Station, NJ) protects against HPV types 6, 11, 16 and 18, and the other vaccine, Cervarix® (GlaxoSmithKline, Rixensart, Belgium) protects against HPV types 16 and 18. HPV.6 and HPV-11 are low-risk virus types that cause approximately 90% of genital warts. Several randomized placebo-controlled trials in human volunteers demonstrated that these prophylactic vaccines elicited high levels of neutralizing antibody and significantly reduced the incidence of persistent HPV-16 and HPV-18 infections and associated moderate-to-high grade cervical intraepithelial neoplasia CIN2/3.
[37-39]
Followup for 4.5 years demonstrated 100% efficacy in the prevention of HPV-16- and HPV-18 CIN2/3 and persistent infections among subjects that were strictly adherent to the study protocol.
[82] Prophylactic vaccination against high-risk HPV types will eventually prevent a significant number of cervical carcinomas, although owing to the prolonged course of cervical carcinogenesis, reduction in cancer incidence will not be profound for several years. These vaccines might also reduce the incidence of HPV-associated oropharyngeal cancers, as well as cancers of the anus, vulva, vagina, and penis. Indeed, the presence of HPV-16-DNA in a relatively high fraction of HPV-associated HNSCC suggests that the currently available vaccines may be particularly effective in preventing this disease.
Despite the promise of these vaccines, there are several issues that still need to be addressed. First, the duration of protection is unknown. Second, virus-like particle vaccines are relatively expensive, and vaccine delivery in the developing world will be difficult. Third, the vaccines will only protect against the highrisk HPV types that the vaccine targets (i.e. HPV-16 and HPV.18) and women must, therefore, continue to undergo Pap smear screening, because they could still be infected by high-risk HPV types that are not included in these vaccines. Finally, these prophylactic vaccines are likely to provide limited benefits to women already persistently infected with high-risk HPV.
[82]
Therapeutic HPV vaccination strategies are also being developed. The high-incidence of cervical cancer in immunosuppressed individuals suggests that cellmediated immunity has a major role in controlling cervical carcinogenesis. The generation of a cell-based immune response to E6 and/or E7 might, therefore, result in destruction of HPV-associated precancerous and cancerous cells that express these viral proteins. Various therapeutic vaccination strategies can elicit a cytolytic CD8 + T-cell response directed against high-risk HPV-E6 and/or HPV.E7 oncoproteins in humans and mice.
[83] In mice, this practice leads to a decrease in the growth of transplantable tumors expressing these viral antigens.
[84] This approach, however, has not yet been successful against CIN and cervical carcinoma in humans.
[85]
An improved mechanistic understanding of the virologic basis for cervical and some head-andneck and other anogenital cancers will help to prevent and treat these diseases. The widespread use of prophylactic HPV vaccines combined with vigilant Pap smear and HPV-DNA testing has the potential to eliminate many of these cancers. Molecular classification of tumors is likely to provide important new information that will allow a better estimate of prognosis and may well influence treatment decisions. In the future, antiviral pharmaceutical approaches and therapeutic vaccination may allow effective, nontoxic therapy. We are optimistic that the tools to control these cancers will soon be available.


High-risk human papillomaviruses encode E6 and E7 oncoproteins that bind and degrade p53 and pRb tumor suppressors, respectively

HPV-associated cancers maintain and express the HPV viral genes even in advanced stages of disease, and repression of viral oncogene expression can prevent the growth and survival of cervical cancer cells

The annual incidence of oropharyngeal cancer in the US increased between 1973 and 2001 in younger individuals, in parallel with an increase in sexual practices that are typically associated with the sexual transmission of HPV

HPV-16 is the most prevalent genotype in cervical carcinoma, and is also the most frequently detected HPV type in HNSCC, found in up to 90% of HPV-positive cases

HPV-associated oropharyngeal squamous-cell cancers have a better prognosis than HPVnegative tumors

Prophylactic vaccination against high-risk HPV types will eventually prevent a significant number of cervical carcinomas and possibly HNSCC, although owing to the prolonged course of carcinogenesis, the reduction in cancer incidence will not be profound for several years
Charles P Vega, University of California, Irvine, CA, is the author of and is solely responsible for the content of the learning objectives, questions and answers of the Medscapeaccredited continuing medical education activity associated with this article.
Research in the authors’ laboratories was supported by grants from the NIH (CA16038) and the Virginia Alden Wright Fund.
Amanda Psyrri, Departments of Medicine/Oncology, Yale Cancer Center, Yale University School of Medicine, PO Box 208020, New Haven, CT 06520-8020,
USA diamando.psyrri@yale.edu
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