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Bast RC Jr, Kufe DW, Pollock RE, et al., editors. Holland-Frei Cancer Medicine. 5th edition. Hamilton (ON): BC Decker; 2000.

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Holland-Frei Cancer Medicine. 5th edition.

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Chapter 20Papillomaviruses and Cervical Neoplasia

, MD and , MD.

In recent years, human papillomaviruses (HPVs) have become the principal focus of efforts to implicate a transmissible virus in the genesis of lower genital tract neoplasia. An explosion in technology has dictated both the tempo and direction of this research, which began with descriptive and experimental pathology, progressed to molecular biology, and finally involved molecular immunology in efforts to both implicate the virus directly in producing neoplasia and unravel the mechanisms of host response.

Biochemical work by several laboratories has unveiled potential mechanisms by which HPV infection may produce neoplastic transformation. Studies with keratinocyte cultures, in which features of HPV-related neoplasia have been reproduced,1,2 have put this information into morphologic perspective. Further, direct analysis of HPV nucleic acids in clinical material has identified the nature of HPV expression and provided morphologic clues as to why certain HPV types may be associated with cancer. Clinical application of this information has been attempted, principally on the basis of the strong association between HPV and cancer. Unfortunately, the strict association between HPV nucleic acids and cervical cancer has been hampered by the discovery of latent or occult virus infection, which, in turn, has complicated the picture of a diagnostic molecular test that would highlight women at risk for developing cancer. Finally, the advent of molecular immunology has produced sobering observations, balancing the hope for a serologic test for HPV exposure with the reality that HPV infection (or exposure) is extremely common, whereas cervical cancer is not. This discovery has been accompanied by a shift in emphasis toward the use of immunology to prevent (by use of vaccines) rather than detect HPV-related disease. This chapter details the molecular basis for HPV-related precursor diseases of the cervix, balancing this information with the morphologic and clinical perspectives that are integral to the management of these extremely common disorders.

Definitions, Mechanisms, and Pathobiology of Genital HPV Infection

Definition of Infection

Genital “infections” are best defined by the presence of clinically or colposcopically identifiable flat or raised lesions that contain papillomaviral DNA, the prototype of which is genital warts. In this instance, infectious virus is likely to be identified within the epithelium (Fig. 20.1A). More recently, the term “infection” has been expanded to include HPV-related precancerous lesions, or even cancers, the term being used loosely to denote the presence of viral DNA. However, virions are less likely to be identified in these processes (Fig. 20.1B).3 As will be detailed subsequently, HPV DNA may be associated with occult viral infection, active infection, or advanced neoplasia (Table 20.1).

Figure 20.1. Histopathology of a classic human papillomavirus (HPV) infection (condyloma) of the cervix associated with low-risk HPV types (HPV types 6 or 11).

Figure 20.1

Histopathology of a classic human papillomavirus (HPV) infection (condyloma) of the cervix associated with low-risk HPV types (HPV types 6 or 11). A. Morphologic features of HPV infection include nuclear atypia in the superficial epithelial cells with (more...)

Table 20.1. Definitions.

Table 20.1

Definitions.

The hallmark of HPV infection is a morphologic transformation of the target tissue. This is not synonymous with the term “transformation” as classically applied to changes in cultured cells produced after introduction of HPV nucleic acids. Rather, it defines the morphologic alterations that can be most consistently associated with the presence of HPV nucleic acids. Depending on the host response and HPV type involved in the infection, it may be defined as a low- or high-grade genital precancer, either of which is distinct from the normal epithelium (Figs. 20.1 and 20.2).

Figure 20.2. Histopathology of cervical intraepithelial neoplasm associated with high-risk HPV types (i.

Figure 20.2

Histopathology of cervical intraepithelial neoplasm associated with high-risk HPV types (i.e., 16, 31, 33, 35, and so on). A. Lesion involving the superficial and crypt (gland) epithelium (large arrowhead). Koilocytotic atypia is present (upper right (more...)

Mechanism of Infection

Papillomaviruses are epitheliotropic, circular, double-stranded DNA viruses that infect the squamous epithelium. The interval from exposure to the development of a lesion varies from a few weeks to several months, and perhaps longer.4,5 It is presumed that the virus gains access to the cervix or lower female genital tract through defects in the epithelium that expose the basal epithelial cells to virion particles. In support of this hypothesis are the demonstration of papillomavirus DNA and RNA in basal cells and the observation that experimental infection of the squamous mucosa by HPV is enhanced by disturbing the epithelial surface (and hence exposing the basal cells) prior to exposure.6 Infection most likely occurs via the receptors in basal cells known as integrins. As the cells containing the viral DNA approach the upper layers of the epithelium, the virus replicates and assembles into virions, which can be detected by electron microscopy or immunohistochemistry (see Fig. 20.1).7 Some of the superficial cells in the infected epithelium characteristically display enlarged, hyperchromatic nuclei, with or without cytoplasmic halos (koilocytotic atypia), and the mature virus usually concentrates in this cell population (see Fig. 20.1) .7,8 Whether koilocytosis is due exclusively to viral replication is controversial, principally because this cytologic phenomenon may exist in the absence of abundant capsid proteins or virions. The implication is that the nuclear hyperchromasia of koilocytotic cells signifies host DNA replication occurring in concert with viral replication.7,8 Although the genital squamous epithelium appears to be the principal site for HPV infection, there is evidence that infection may occur in germinal or undifferentiated epithelial cells that give rise to both the squamous and glandular components of the cervical mucosa. HPV nucleic acids have been isolated from neoplasms not clearly derived from squamous-committed epithelial cells, most notably adenocarcinomas and undifferentiated carcinomas (small cell carcinoma).9,10

Specificity

The squamous epithelium is most susceptible to HPV infections. In particular, squamocolumnar junctions, where the glandular portion is undergoing replacement or transformation by the squamous epithelium (transformation zones), are most vulnerable to the genital papillomaviruses.11 These transformation zones contain stem cells that may give rise to both squamous and columnar epithelia. Infection with “genital types” has been demonstrated in other mucosal sites in which this process of epithelial transformation takes place, including the larynx,12 oropharyngeal mucosa,13 anus,14 esophagus,15 subungual mucosa (nail bed),16 and conjunctiva.17 Kreider and colleagues demonstrated that some of those sites are particularly vulnerable to experimental infection with genital viruses.18 This indicates that the genital HPV types require specific conditions provided by certain locales for infection to occur, or characteristics facilitating morphologic transformation once infection has taken place. One component of this equation is the differentiating capacity of the infected stem cells.19

HPV and Human Genital Neoplasia

Evolution of the Concept

Although studies with animal papillomaviruses established their potential role in the genesis of neoplasia, the most significant link between HPV and human cervical neoplasia came in the form of observations that koilocytotic atypia, a common cytologic feature of abnormal Papanicolaou smears, was a cellular marker for the presence of genital HPV infection.8,20,21 By virtue of its high frequency, this cytologic abnormality focused researchers on this virus and its association not only with genital warts, but also with cervical precancerous lesions (cervical intraepithelial neoplasia [CIN] or cervical dysplasia). Thus, the initial hypothesis that HPV was an oncogenic virus in the cervix was derived not only from molecular biology but also from morphologic evidence via the association between genital papillomaviruses, abnormal Papanicolaou smears, and cervical precancers.5 The cloning of genital HPVs redirected attention from the morphology of HPV infection to the molecular pathology of HPV-related diseases, in that molecular probes could identify HPV nucleic acids in the absence of viral particles or capsid proteins. Thus, it became possible to identify HPV nucleic acids not only in condylomata, but also in squamous precancers and carcinomas of the female genital tract.22–25 As part of this progression, the discovery of a variety of different HPV types laid the foundation for establishing that specific HPV types are associated with certain types of genital lesions.26 Currently, over 60 distinct types of HPV have been identified, many of which are associated with specific clinical and pathologic characteristics.23 For example, genital warts and condylomata are associated with certain viral types (types 6, 11, and others), whereas precancerous lesions (CIN) and invasive cancer are frequently associated primarily with types 16, 18, 31, 33, 35, and others (Table 20.2).23 In essence, the “higher-grade” precancers are more likely to harbor “high-risk” HPV types, implying that, as a group, these lesions are more likely to progress to carcinoma, if not treated (see Fig. 20.2). The association of high-risk HPV types with both high-grade precursors and cancers has strengthened the hypothesis that infection by specific types produces specific kinds of precursor lesions that may evolve into carcinoma, depending on host factors.22,24,27 One interesting departure from the above concept occurs with HPV-18, which is infrequently associated with squamous precursors and more frequently associated with invasive squamous, glandular, and undifferentiated cervical cancers.9,10,28,29 However, in precursor lesions, HPV-18 is frequently identified in lesions of lower-grade morphology, in contrast to the typical high-grade intraepithelial lesions associated with HPV-16.30 The bland morphology of many HPV-18–related precursors contrasts with the high-grade morphology of HPV-18–associated cancers, but this difference cannot be linked to functional differences in sequences encoding in vitro transforming potential, transcriptional regulation, or transactivation functions.

Table 20.2. Most Common Genital HPV Types.

Table 20.2

Most Common Genital HPV Types.

Molecular Basis For HPV-Related Neoplasia

The molecular basis for papillomavirus effects on host squamous cells is based on and supported by the following observations:

1.

Lesions associated with high-risk HPV types frequently possess morphologic and biologic characteristics that distinguish them from infection by other HPV types, suggesting that molecular events occur during infection that are unique to these virus–host relationships.26,27,31,32 For example, HPV-16–related precursors produce fewer virions, are associated with greater cytologic atypia, and, by inference, frequently contain aneuploid cell populations (see Fig. 20.2).31,32 The supposition is that some component of infection by this and similar viruses causes fundamental changes in the biology of the epithelium, which, in turn, increases the risk of persistence of morphologic abnormalities and, in some cases, the risk of progression to cancer.

2.

HPV types associated with neoplasms (high-risk HPV types) differ from low-risk HPV types in molecular sequence and in the effects of these sequences on cells. Clues to what makes HPV-16 infection unique vis-à-vis the so-called low-risk (HPV-6) infection have been forthcoming from several lines of investigation, all of which center on the viral genome itself (Fig. 20.3).33 Mechanisms that have been studied and that may distinguish low- from high-risk viruses include (1) differences in the expression of the so-called transforming genes, such as the E6/E7 and E5 oncoproteins; (2) the process of genomic integration; and (3) mechanisms by which the upstream regulatory region is influenced by exogenous factors, such as receptor complexes. These mechanisms are summarized in Figure 20.4.34–38

Figure 20.3. Schematic of the HPV-16 genome, outlining potential “genes” (open reading frames) and their possible functions.

Figure 20.3

Schematic of the HPV-16 genome, outlining potential “genes” (open reading frames) and their possible functions. ORF = open reading frame; URP = upstream regulatory region.

Figure 20.4. Schematic of potential mechanisms of HPV-related neoplastic transformation.

Figure 20.4

Schematic of potential mechanisms of HPV-related neoplastic transformation.

Most of what is known about HPVs is derived from analogous studies with bovine papillomaviruses (BPVs). Lowy and colleagues established that 69% of the BPV genome could alter the growth characteristics of cells in culture (transformation).39 Subsequent sequencing of this viral DNA and HPV viral DNA established that both human and animal HPVs share similar genomic organization in which the region corresponding to the transforming region of BPV is designated as the early (E) region. In contrast, the late (L) region encodes capsid proteins and does not possess transforming potential.23,37 Studies of cell transfection and in vitro biochemical assays combined with mutational analysis have identified specific open reading frames (ORFs) that produce gene products (proteins) possessing different biologic properties (see Fig. 20.3).23 The functions of the early regions of the HPV genome and their influence on carcinogenesis have recently been delineated through structure-function analysis of these ORFs within the different viral types.40 Two genes that are actively expressed following viral integration, the E6 and E7 ORFs, are the major mediators of cellular transformation. This capability relates to their unique abilities to bind and block the function of critical cellular growth regulatory proteins. The E7 ORF encodes a 21-kd nuclear phosphoprotein that is able to cooperate with ras to transform primary rodent cells.30,41,42 Similar to the large T antigen of SV40 and the E1A protein of adenovirus, the E7 protein is able to bind the retinoblastoma (RB) gene product.6,40 This binding capability of E7 for RB differs between the different groups of HPV, in that the oncogenic viral types (HPV types 16 and 18) have a greater binding affinity for this regulatory protein than do the nononcogenic types (HPV types 6 and 11).43 Further, the efficiency of binding correlates with the transforming capacity of these viral types.44 Binding affinity appears to be influenced by a single amino acid difference within the E7 ORF, a difference that is consistently noted between the viral types.44 Insight into the effect of this protein interaction has also been achieved. Binding of RB by the E7 protein has been shown to release other cellular factors such as the E2F transcriptional factor.6,40 Increased intracellular concentrations of free E2F may result in increased cellular proliferation, a possible mechanism for the transforming potential of HPV E7.

The product of the E6 ORF also has important functions in cellular transformation. Like E7, the E6 protein displays characteristics similar to SV40 and adenovirus by binding p53, a key cell cycle regulatory protein.5 p53 is known to block G/S-phase cell cycle progression following DNA damage, allowing repair to take place before mitosis can resume.45 Mutations in p53 that result in loss of functional protein are among the most common genetic aberrations present in solid tumors.45 However, in HPV-positive tumors, p53 mutations are uncommon and p53 levels are nearly undetectable.46 It appears that by binding p53, E6 is able to induce proteolysis of this protein via the ubiquitin pathway,47 effectively removing the p53-mediated block to cellular proliferation. Other perturbations of the cell cycle mediated by the E6/E7 oncoproteins may take further advantage of the absence of p53 function.

Another ORF with potential transforming ability is the E5 ORF, although the evidence in support of this is limited principally to the BPV system.48 The relationship of this protein to human disease is unclear. Its transforming potential relates to the ability of E5 to bind intramembrane proteins, such as a 16-kd component vacuolar ATPase,49,50 although the mechanism of this interaction is unknown. E5 also appears to modify the internalization and phosphorylation of certain growth factor receptors, growth factor (EGF) and colony-stimulating factor (CSF)-1.50 A final region that may distinguish high- from low-risk HPV is the upstream regulatory region (URR), or long control region (LCR). This enhancer within the URR activates expression of the transforming regions of the HPV genome, E6, and E7.6,40,51 During viral integration into the host genome, the E1 or E2 region is consistently disrupted.51 Loss of these ORFs leads to derepression of the URR enhancer, and the E6/E7 transforming proteins are expressed. Integration of the virus and interruption of E1/E2 appear to be important steps in cervical carcinogenesis. This region contains sequences that bind nuclear proteins and that contain glucocorticoid receptor sequences that will enhance transcription in a variety of HPV types when exposed to glucocorticoids. Studies by Pater and colleagues demonstrated that dexamethasone is required for oncogenic transformation of cultured cells by HPV-16 DNA and the ras oncogene, and that this phenomenon is not reproduced with HPV-11.52 This is of particular interest in light of epidemiologic studies associating oral contraceptive use with the risk of cervical cancer.53

Other ORFs include those encoding capsid proteins (L1, L2) of unknown function, such as the E4 ORF.54 The latter is produced in abundance in some HPV infections.27,55–57 In addition, the E2 ORF encodes an important product that both positively and negatively regulates the upstream regulating region.58 Finally, the intact E1 ORF is required for maintenance of the plasmid state, which perhaps explains why it is the site of interruption when genomic integration takes place.1 In recent years, the experimental infection of cervical grafts with HPV-11 has produced genital warts in nude mice.59 Moreover, transfection of human keratinocytes with HPV DNA has verified the necessity of the E7 ORF in the transformation process, and demonstrated that HPV-16 alone will produce an aneuploid cell population with many characteristics of a precursor lesion.60 Co-transfection of HPV-16 DNA with oncogenes has likewise produced similar lesions and, in some studies, neoplasms with metastatic potential.61 What has not been accomplished has been the successful completion of the life cycle of the virus in tissue culture or the production of infectious virus from cells into which DNA alone has been introduced. These remain the principal obstacles to successfully mimicking in vitro the in vivo state of the virus, as well as manipulating the viral genome to identify the critical components of infection.

Models for the putative functions of the above ORFs in precursor and cancer development include the following: (1) function of the E6-E7 domain is virtually always present in tumors, via transcription of either episomal or integrated sequences; (2) transfection of keratinocytes with the HPV-16 E6-E7 sequences produces proliferations resembling high-grade cervical intraepithelial neoplasia (CIN); (3) preservation of vegetative functions invariably segregates with low-grade CIN; (4) high-risk HPV types, such as HPV-16, are infrequently associated with abundant vegetative functions, suggesting a lack of efficient replication and viral assembly associated with this virus; (5) HPV-18–associated low-grade CIN frequently produces abundant capsid proteins, much as low-risk HPV types do, implying that maintenance of vegetative functions will suppress effective expression of the HPV-18 oncogenes. In contrast, HPV-18–associated cancers invariably harbor integrated sequences. Thus, HPV-18 is a model of an HPV with powerful in vitro immortalization potential that is abrogated in vivo, provided that vegetative functions are preserved. Determining which factors influence these differences, including host functions, may provide clues to the role of host susceptibility in this disease.

Occult Infection

Considerable evidence has accumulated identifying HPV DNA in tissue or cell preparations that do not exhibit significant morphologic abnormalities. The basis for the hypothesis that clinically occult HPV infection exists has been established previously, if simply from the observation that new disease may occur in sites where previously there had been no lesion. In the first molecular analysis of this phenomenon, Steinberg and colleagues reported finding HPV DNA sequences in normal-appearing laryngeal mucosa from patients with a history of laryngeal papillomas but who were at the time in apparent remission.62 Ferenczy and colleagues linked occult infection to clinical disease in their study of patients with vulvar warts or precancers who were undergoing laser therapy. They found that grossly normal squamous epithelium adjacent to the treatment field often contained HPV DNA, and that patients with this clinically “occult” infection had a higher frequency of recurrences than those who did not.63 This finding is reinforced by observations that warts may preferentially occur at sites of trauma, emphasizing the relationship between healing and viral activation.64

The studies described above addressed populations with documented HPV-associated lesions either concurrently or in the past. It is possible that despite appearing normal, the tissue contained HPV DNA because of its proximity to the tissue clinically infected by the virus or from shed cellular material (contamination) in adjacent lesions. Whatever the mechanism, the important questions to be addressed are whether it occurs in women with no history of HPV infection or abnormal Papanicolaou smears and, specifically, if it has prognostic importance.

Numerous studies have reported the detection of HPV DNA in women with no history of previous HPV-related disease.65 The detection rate of HPV in asymptomatic women varies according to age and sexual activity. HPV positivity has been correlated with lifetime number of sexual partners;66 however, other correlates include other genital infections, frequency of sexual contact, lack of use of barrier contraceptives, and number of sexual partners in the recent past. The last parameter underscores the influence of recent rather than remote sexual contact on detection of HPV DNA.

Estimates of positivity have varied according to age and the above factors. Rosenfeld and colleagues observed a rate as high as 39% by Southern blot analysis in sexually active adolescents.67 The rates in clients of sexually transmitted disease clinics are also high. In contrast, the rates in older women (ages 35–55 years) have been sharply reduced, measuring approximately 2 to 3%.53 In a recent study of women undergoing routine hysterectomy, we observed an index of 2.1% and a rate of high-risk types of less than 0.25% in middle-aged women (unpublished observations).

The precise location of HPV DNA sequences in normal squamous epithelium remains unknown. Numerous studies using relatively sensitive techniques such as in situ hybridization, have, with rare exceptions, failed to localize HPV nucleic acids in the normal epithelium, despite the confirmation on Southern blot hybridization.42 This does not necessarily exclude the potential importance of these sequences, in that Nuovo and colleagues found that a large proportion of HPV-related lesions contained more than one HPV type when analyzed by polymerase chain reaction, despite the fact that only one HPV type could be detected by in situ hybridization.68 This suggests that when a lesion develops from infection by a single virus type, other virus types in the vicinity are in some way inhibited from co-infecting or producing morphologic changes. In fact, the frequency of histologically demonstrable double infection is less than 5%.68 Nevertheless, Nuovo and colleagues demonstrated that recurrent lesions following ablation were frequently associated with HPV types other than the original.69 Although the role of occult infection in these recurrences is unknown, this and other findings suggest that occult infection may have clinical significance under certain circumstances.

Until recently, the predictive value of HPV DNA positives was poorly understood. Lorincz and colleagues did not correlate HPV DNA with a high risk of disease in the absence of clinical (or Papanicolaou smear)-findings.59 However, Koutsky and colleagues, in a study of women in a sexually transmitted disease clinic, observed that 28% of HPV-positive women developed a CIN lesion within 2 years, versus only 3% of HPV-negative women.70 Subsequent studies by Koutsky and colleagues established that many HPV infections, if followed up closely, will manifest cervical abnormalities (L. Koutsky, personal communication). On the basis of the high incidence of abnormal Papanicolaou smears in younger women and the disproportionate number of HPV positives in this population, the model for HPV infection includes a high rate of acquisition at a young age, transient infections/lesions in many women, the development of immunity, and a low rate of lesion detection/HPV positivity in women over age 35 years. A proportion of high-risk HPV types will produce high-grade CIN lesions that will persist and presumably constitute a risk factor for progression to invasive carcinoma.

Applications To Clinical Medicine Background

The prevention of cervical cancer is based on the Papanicolaou smear. Because the majority of cervical cancers are preceded by a cervical precursor (CIN) lesion, often by many years, the detection of these precursors is fundamental to cancer prevention. Precursor lesions are recognized clinically on colposcopy, where precursor lesions can be identified following the application of acetic acid. The use of colposcopy has maximized the targeting of lesions for biopsy, and outpatient removal is the usual approach, including cryotherapy, laser, and, recently, loop electrical excision.71 The latter procedures target the entire transformation zone, removing the lesion and replacing the process of chronic repair with a brief period of re-epithelialization.

Diagnostic Classification

Because high-risk HPV types are strongly associated with squamous cell carcinomas of the cervix, efforts have been made to refine diagnostic criteria that would be most likely to identify an infection with such types. These efforts have centered on distinguishing HPV infection alone (cytopathic effect) from the more pronounced features associated with high-risk HPV types—dysplasia. In essence, CIN has been redefined in practice, with CIN I corresponding to lesions closely resembling condyloma, CIN II as lesions classically called dysplasia, and CIN III as lesions previously termed carcinoma in situ.72

Clinical Management

Because lesions in the CIN I category are least likely to progress to carcinoma, recent consensus conferences have proposed that patients whose Papanicolaou smears contain the features of condyloma or CIN I be followed up by repeat smear alone.73 This is supported by the fact that 80 to 90% of smears exhibiting these features will be associated either with CIN I or with lesser (negative) changes on follow-up biopsy.74 It is generally accepted that the risk of following higher grade abnormalities (CIN II–III) is unacceptable, notwithstanding the fact that few cases will progress to carcinoma over short-term follow-up.75

An important, albeit controversial, issue concerns the value of HPV testing in the management of women with suspected cervical abnormalities. Although HPV DNA testing is considered an imperfect alternative to the Papanicolaou smear in the prevention of cancer, a significant proportion of cancer cases do develop despite screening, in addition to about one third that develop in women who have never been screened.46 Here, the most compelling use of HPV testing would be for identifying women at risk for cervical neoplasia, and follow-up studies of women who harbor high-risk HPV types in the genital tract indicate that these individuals have a greater than 10-fold risk of developing a high-grade precancer.76,77 However, the high frequency of HPV positives in young women, the fluctuation in HPV status in these women, and the inherent high risk of young women developing preinvasive cervical disease leaves the practical value of this information uncertain.70,78 Nevertheless, the information is a compelling endorsement of the concept that exposure to HPV increases risk of developing a precancerous cervical lesion.

The strongest argument for HPV testing has been its application to the management of women with Papanicolaou smear abnormalities that are not diagnostic of squamous precursor lesions, but fall out of the “comfort zone” of benign cellular changes.79 These nondiagnostic squamous atypias or “atypical squamous cells of undetermined significance” (ASCUS) are emblematic of how introduction of a particular diagnostic term or language into routine cytopathology practice alters patient management.80 By definition, a diagnosis of ASCUS implies diagnostic uncertainty and inherently has poor reproducibility between cytopathologists. For this reason, ASCUS has been proposed by some as a suitable target for HPV-directed triage.79 The commitment to this concept is of such magnitude that a large National Cancer Institute (NCI)– sponsored study has been initiated to explore the role of HPV testing in the triage of ASCUS.

At present, it is clear that women with ASCUS who are HPV positive have a 10-fold higher risk of a biopsy proven dysplasia than HPV-negative individuals (50 vs. 5%).79 For this reason, it may be argued that HPV testing will help enrich the population of women with smear abnormalities for those with the greatest risk of a positive follow-up biopsy. It should also be noted that within the spectrum of ASCUS, subsets with inherently low risk of a positive biopsy can be identified, albeit less objectively, by morphologic approaches.41 Studies of women with ASCUS smears who are postmenopausal or those studies accompanied by reactive cellular patterns show these groups to be at a very low risk of either concomitant HPV positivity or follow-up abnormalities on biopsy. 41,81 Nevertheless, HPV testing remains the most objective triage method for selecting women with ASCUS who are at risk for subsequent disease.

In general, more thorough health-care delivery following Papanicolaou smear screening is the most effective alternative, although it is conceivable that molecular testing of certain populations with a low background index of HPV DNA (such as older women) could provide information that would augment the information from conventional Papanicolaou smear screening. However, women who do not have access to a gynecologic examination will, by definition, benefit neither from a Papanicolaou smear nor from viral testing as conventionally applied.

At present, the importance of focusing on sexual transmission is unclear. Male sexual partners carry the viruses but, in general, exhibit no clinical disease, or only subtle infections on the penile shaft, scrotum, and urethral meatus. Efforts to detect and eradicate disease in this group have been encouraged, but the actual impact of this approach on the cancer incidence rates is unclear, as is the importance of benign genital warts on areas less susceptible to neoplastic change, such as the vulva and vagina. The failure of high-technology therapy (e.g., laser therapy) to eradicate these infections, much less latent virus infection, has encouraged a more conservative approach to generic HPV infection and focused efforts on identifying subsets of women who are at greater risk. This group includes African Americans, smokers, and individuals who have disease on the cervix as depicted by clear-cut Papanicolaou smear abnormalities.65

Prevention

Excluding barrier methods of contraception, Papanicolaou smear screening, and HPV DNA testing, prevention of HPV-related cervical neoplasia will depend on whether these disorders can be prevented by vaccines. Concerning the immune response, the most promising studies are in the field of vaccination. Because papillomaviruses cannot be grown in culture, the study of their immunogenicity has been limited previously to serologic studies using denatured target peptides generated by recombinant technology (Fig. 20.5).82 In contrast, the most likely targets are conformational epitopes on the surface of the capsid. Recently, a number of investigators have succeeded in producing intact capsid particles by expressing the entire late region of papillomaviruses in baculovirus vectors or other eukaryotic systems. These empty capsids contain the conformational epitopes felt to be operative in generating host immunity, and their study provides the opportunity to manipulate the viral genome to produce reagents that can be used to study (or generate) host immunity.83 This avenue of investigation is, at present, the most promising because it offers the advantage of intact particles that are highly immunogenic and focuses on the capsid proteins, which are likely the first to be detected by the host immune system. Moreover, vaccination with structural components of the virus avoids the obvious concerns attendant on using proteins with known transforming potential in vitro. The major question will be whether systemic immunization will provide lasting protection to local mucosal sites, such as the cervix, or whether novel delivery systems will be required.

Figure 20.5. Immunoblot (Western blot) with human serum demonstrating seroreactivity to an in vitro synthesized pATH fusion protein containing HPV-16 L2 (capsid) protein.

Figure 20.5

Immunoblot (Western blot) with human serum demonstrating seroreactivity to an in vitro synthesized pATH fusion protein containing HPV-16 L2 (capsid) protein. The sera react with the fusion protein (86 kd) in lane b (arrowhead). Lane a, containing a vector (more...)

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