Studies have shown a strong association between certain human papillomaviruses and the development of cervical carcinoma and its precursor lesions. The oncogenic potential of papillomaviruses has been clearly demonstrated in both laboratory animals and cultured cells. Recent advances in our understanding of viral pathogenesis have provided insights into the natural history of papillomavirus infection and subsequent development of neoplasia. A more thorough understanding of the molecular mechanisms responsible for viral oncogenesis will facilitate the development of novel preventive and therapeutic strategies to prevent and treat papillomavirus-associated cervical neoplasias. Strategies under current investigation are focusing on the induction of effective humoral and cell-mediated immunity, the expression of HPV gene products, and cofactors that interact with HPV gene products to affect cell transformation. As a result of these investigative efforts, prophylactic HPV capsid vaccines and other gene therapies may soon become clinically available.
Papillomaviruses are ubiquitous in a wide variety of vertebrate species, including humans. They infect and cause proliferative lesions of cutaneous and mucosal squamous epithelium . More than 60 types of human papillomaviruses (HPVs) have been described (Table 1) [2,3], each of which shows a particular predilection for tissue sites and has defined oncogenicities. Particular interest has focused on HPVs as-sociated with anogenital tract disease. There is a well-established association between HPV infection, cervical dysplasia, and cervical carcinoma [4-6].
In 1994, 15,000 new cases of invasive cervical cancer and 4,600 deaths attributable to cervical cancer are projected in the United States.7 Statistics on cervical cancer worldwide are much more staggering, with 500,000 deaths per year caused by this cancer . These figures highlight the public health significance of controlling cervical cancer and its precursor lesions.
Recent advances in immunology and molecular biology have broadened our understanding of the biology of HPV. In particular, insights into the molecular mechanisms of HPV-mediated tumorigenesis may be the basis for new preventive and therapeutic strategies for HPV-associated disease (Figure 1).
The viral etiology of warts was first described by Licht in the late 19th century . In 1933, Shope and Hurst described the first DNA tumor virus isolated from the papillomavirus of cottontail rabbits . Condyloma acuminata, long recognized to be sexually transmitted, were linked to a viral etiology when virus particles were detected in genital warts by electron microscopy in the 1970s [10,11].
Papillomaviruses have been classified into the papovavirus group because of similarities among papillomavirus, polyoma virus, and the vacuolating virus of monkeys . The structure and genetic organization of all the papilloma- viruses are strikingly similar. Papillomaviruses consist of a 55-nm, nonenveloped, icosahedral-shaped virion whose genome is organized as closed, circular, double-stranded DNA of approximately 8,000 base pairs in length  (Figure 2).
The papillomavirus genome can be divided into three functional regions (Table 2) : The "early" region contains eight open reading frames, or genes, whose products are responsible for viral DNA replication, transcriptional control, and cellular transformation. The "late" region encodes the two structural capsid proteins, L1 and L2, of the virion. The "long control region" contains the origin of DNA replication, promoter elements, and transcriptional enhancer sequences.
The true prevalence of HPV infection in the general population is unknown. This is due to a number of variables, including coital activity, host response to infection, and the diagnostic modality used to detect HPV infection. Cutaneotropic HPVs cause verruca plana, which is common among young children, and verruca vulgaris, which is common among adolescent children . Mucosotropic HPVs produce a variety of lesions of the conjunctiva, oropharynx, and larynx, in addition to anogenital tract lesions.
Sexual transmission of anogenital warts is supported by data confirming the presence of similar HPV types on cervical and penile lesions of sexual partners . Several factors affect the rate of transmission of mucosotropic HPVs. These include coitus with multiple sexual partners, immunodeficient states, and pregnancy.
These highly infectious viruses have a relatively long incubation period following inoculation. Lesions caused by mucosotropic HPV types usually appear within 4 to 6 weeks in humans. The same incubation time is observed with mucosotropic viruses that infect keratinocytes in the athymic nude mouse
The host response to infection is similar for all HPV types. All three types of squamous epithelia (cutaneous-keratinized, mucosal-nonkeratinized, and metaplastic) are susceptible to HPV infection. While infection may manifest itself differently for different HPV types, infection begins in the basal layer of squamous epithelia. Presumably, virus from infected cells is released into epithelial breaks of the susceptible host .
Human papillomaviruses demonstrate specific tissue tropism to anatomic sites. Equally important is the fact that HPVs can only synthesize structural proteins that encapsidate the genome and form virions in the most differentiated keratinocytes.
Three Sequelae of Infection
Following infection with HPV, three sequelae are possible:
First, the HPV genome can stabilize as a nonintegrated episome and remain latent in the host without producing clinical or morphologic changes in the squamous epithelium.
Second, active infection can be established with vegetative replication of HPVs, which induces the proliferation of squamous epithelia into benign tumors (warts, papillomas).
Third, the HPV genome can become integrated into the host genome, which interrupts its control of oncoproteins of highly oncogenic viruses.
Expression of early and late viral gene products accounts for the morphologic changes seen in affected epithelia. Early gene expression causes cellular proliferation, which results in acanthosis. Late gene expression results in production of viral capsid proteins, which are evident (by electron microscopy and immunocytochemistry) only within nuclei of terminally differentiated, superficial epithelial cells (keratinocytes). In latently infected cells and benign tumors, the HPV genome is present in nonintegrated, episomal form. Viral capsid assembly in productively infected, terminally differentiated keratinocytes causes degenerative changes in the nuclei and cytoplasm, which are recognized histologically as koilocytosis .
Latent infections with no pathologically identifiable lesions comprise a large reservoir of virus that may be reactivated for transmission and autoinfection. It is believed that 10% of sexually active individuals harbor latent HPV infections . Active infections occur in up to 5% of sexually active women, and appear as flat or exophytic condylomas of the cervix, vagina, or vulva. Condyloma acuminata are usually associated with HPV types 6 and 11.
Association with Dysplasia and Malignancy
Infection with certain HPV types has a high probability of being associated with dysplasias (types 6 and 11) or with malignancies (types 16 and 18). Squamous intraepithelial lesions show characteristic disorderly, undifferentiated, proliferating basaloid and parabasaloid cells that occupy different portions of the epithelium, from the lower third in mild cervical intraepithelial neoplasia to full epithelial involvement in carcinoma in situ. Conversely, invasive cervical carcinomas extend beyond the basement membrane.
The HPV genome in malignancies is usually not episomal, but rather, is integrated into the host genome in at least 80% of cervical cancer cases. The viral E2 gene serves as the most significant site of integration into the host genome. With integration, normal regulatory function of E2 is interrupted. This event appears to be critical for tumorigenesis. Loss of regulation results in overexpression of viral E6 and E7 oncoproteins, which are known to inactivate the cellular tumor-suppressor gene products, p53 and pRb, respectively .
It is not known which stage of HPV genome integration correlates with the change from dysplasia to malignancy. Integration of the viral genome is not always required for tumorigenesis. In some HPV-16 and other HPV-associated carcinomas, viral DNA exists in an extrachromosomal state .
In vitro biologic assays have enabled investigators to study the transforming activity of cloned human papillomavirus DNA on primary and immortalized cells, and thereby evaluate the role of viral genes in tumorigenesis. Assays of infected and transfected murine and human cells have been important in determining whether continually expressed E6 and E7 are necessary and sufficient for in vitro transformation . Some cell lines produced by these assays are tumorigenic in nude mice alone; others require cooperation with the expressed ras oncogene to become tumorigenic. Tumorigenicity in these instances is dependent upon HPV type .
Host Immune Responses
Immunologic response to HPV infection is an important aspect of host response. Persistence or spontaneous regression of lesions is related to cell-mediated immunity. Patients with altered cellular immunity (immunosuppression, immunodeficient states, pregnancy) have a higher incidence of warts and condylomas . Patients with the congenitally acquired disease of impaired cellular immunity, epidermodysplasia verruciformis, have skin warts and increased rates of detection of HPV types 5 and 8. These warts have a high likelihood of transforming into squamous or basal cell carcinomas .
Finally, HPV-associated primary, metastatic, and recurrent cervical cancers frequently exhibit a reduction in or total loss of allelic expression of critical major histocompatibility complex class I molecules, which are involved in antigen presentation at the cell surface and in antigen recognition. Downregulation of these molecules may enable cervical cancers to escape cell-mediated immune surveillance .
Humoral immune responses to HPV infection have been incompletely quantified. Sera from animals and humans with a history of infection generally react positively to enzyme-linked immunosorbent assays with denatured papillo- mavirus capsid proteins [28-30]. Antibodies are detected in rabbits and mice inoculated with intact HPV virions [28,31]. Humoral immunity appears to protect the host against HPV infection and its transmission.
Recent advances in molecular biology have enabled investigators to synthesize recombinant virus-like particles and capsid proteins of papillomaviruses that react with conformational-dependent, neutralizing antibodies [32-35]. This development will allow for the investiga-
tion of the role of humoral immunity in the natural history of papillomavirus infection.
Oncogenic Potential of HPV Types
In an epidemiologic study of 2,627 women, Lorincz et al examined the prevalence of anogenital HPV infection among normal women and women with premalignant and invasive lesions using southern hybridization. The oncogenic potential of 15 anogenital HPV types was defined. Low-risk HPV types (6, 11, 42, 43, and 44) were found in 20% of low-grade squamous intraepithelial lesions, and were absent in all cancers. Intermediate-risk HPV types (31, 33, 35, 51, 52, and 58) were detected in 23% of high-grade squamous intraepithelial lesions and in 10% of cancers. High-risk HPV types (16, 18, 45, and 56) were detected in 53% of high-grade squamous intraepithelial lesions and in 74% of cancers. Adenocarcinomas of the cervix were usually caused by HPV type 16 or 18.
Continuum of Disease Progression
Studies have documented a continuum of disease progression from squamous intraepithelial lesions to invasive carcinomas. If left untreated, 16% of cases of mild dysplasia will progress to carcinoma within 84 to 96 months, two-thirds will regress, and 22% will persist as mild dysplasia . The majority of high-grade lesions will persist or progress. Two-thirds of cervical intraepithelial stage III lesions will progress to invasive cancer within a mean of 10 years .
Finally, human papillomavirus DNA has been detected, via sensitive polymerase chain reaction assays, in more than 95% of squamous and adenocarcinomas of the cervix . This finding, combined with data showing odds ratios for highly oncogenic HPV types ranging from 31 to 296 for the occurrence of cervical cancer, supports the association of certain HPV infections with cervical neoplasia . Clearly, other factors are probably operative in the development of cervical cancer (ie, tobacco, oncogenes), but HPV is necessary although perhaps not sufficient. Eradication or protection against HPV infection would decrease the number of cervical cancers, perhaps by as much as 95%.
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