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Abstract Cervical infections by approximately 15 human papillomavirus (HPV) types are the necessary cause of cervical cancer and its immediate precursor lesions. However, oncogenic HPV infections are usually benign and usually resolve within 1–2 years. A few of these infections persist and progress to cervical precancer and cancer. A number of cervical factors, such as infection by sexually transmitted pathogens other than HPV, cervical inflammation, and antioxidant nutrients, may influence the natural history of HPV infection along the pathways of persistence and progression or resolution. We examine the possible roles of these HPV cofactors in cervical carcinogenesis and discuss new methodologies that may enable researchers to measure relevant markers of the cervical microenvironment in which these cofactors may be active. Establishing oncogenic human papillomavirus (HPV) as the necessary (1) but infrequent cause of cervical cancer has led to an epidemiologic search for factors that influence the fate of an HPV infection—i.e., factors (HPV cofactors) that might alter the natural history of an HPV infection by increasing the likelihood of viral persistence and progression to cervical precancer (histologic diagnosis of cervical intraepithelial neoplasia [CIN] grade 3 [CIN3]) and cancer above the probability attributable to HPV alone or, conversely, the clearance of an HPV infection. A number of HPV cofactors have been suggested; the most well-known of these factors are smoking, oral contraceptive use, and parity [reviewed in (2)]. Mechanistically, these HPV cofactors may act to increase viral persistence via immune suppression and/or to cause direct tissue damage that increases the predilection for the development of high-grade cervical neoplasia. However, these well-known cofactors do not account for 100% of the cervical cancer cases; thus, there is interest in identifying additional cofactors. A number of less established HPV cofactors, including genital tract infections (e.g., Chlamydia trachomatis and herpes simplex virus type 2 [HSV-2]), cervical inflammation, and nutritional factors, have been suggested based on recent epidemiologic evidence. These cofactors appear to be directly related to the physiologic and immunologic state of the cervix, i.e., the cervical microenvironment. The focus of this chapter will be to examine the potential role of these less well-known cofactors in the outcome of HPV infections and issues related to measuring the effects of these cofactors at the cervix. Cofactors Co-infection In 1989, Schmauz et al. (3) found that multiple sexually transmitted infections (STIs) were a risk factor for cervical cancer, suggesting that additional non-HPV STIs may act as HPV cofactors, even after adjustment for HPV. Since this study, several other studies have assessed the association between STIs and HPV carcinogenesis, primarily focusing on C. trachomatis. One case–control study (4) found that, among oncogenic HPV-infected women, antibodies to C. trachomatis were associated with a twofold increased risk of cervical cancer. A seroepidemiologic prospective study (5) found a 2.5-fold increased risk of cervical cancer associated with C. trachomatis seroconversion for any serovar and a 6.6-fold association with the G serovar, although the conclusions from that study are difficult to interpret because of improper adjustment for HPV and the implausibility of a serovar-specific effect. A recent multicenter case–control study (6) has also provided strong evidence for HSV-2 infection, as measured by seroconversion, as an HPV cofactor. Other non-HPV STIs have not been carefully evaluated as HPV cofactors, although their very low prevalences suggest that their contribution to cervical cancer rates would be minor, even if they were cofactors. Although there is strong evidence that C. trachomatis and HSV-2 infections increase the risk of cervical precancer and cancer, further confirmation is needed. In particular, there is always the concern that infections by these STIs are surrogates for higher risk behaviors that increase the exposure to HPV. Prospective studies of HPV-infected women, with multiple measures of HPV and with DNA measurements of STIs demonstrating concurrent infection, could help to differentiate between STIs as true HPV cofactors or as HPV surrogates. If these co-infections are HPV cofactors, such studies could also be informative about the stage in the natural history of HPV that co-infection with STIs is most likely to influence. Cervical Inflammation Our understanding of the role of STIs in the natural history of HPV is also limited by the lack of understanding of the biologic effect of STIs in the development of precancer and cancer. There are several mechanisms by which these co-infections might act, such as direct genotoxicity, but perhaps the most likely biologic mechanism is the induction of inflammation at the cervix leading to genotoxic damage via reactive oxidative metabolites. For example, C. trachomatis is a well-known cause of cervicitis. Moreover, cervical carcinoma cell lines infected with C. trachomatis secrete greater proinflammatory cytokines than either primary uninfected cervical cells or noncervical epithelial cell lines (7), suggesting that co-infection of HPV and C. trachomatis in cervical tissue may result in a more profound inflammatory state than for C. trachomatis infection alone. HSV-2 can cause periodic ulcerative sores, suggesting that it may also act through an inflammatory pathway, but viral activation or viral shedding has not yet been measured in the context of an HPV infection. Of note, there is no evidence to suggest that HPV infections alone induce an inflammatory state. Inflammation has long been considered to be a risk factor for cancer at several organ sites, and chemoprevention efforts for colon and other cancers are using nonsteroidal anti-inflammatory drugs for targeted inhibition of cycloxygenase-2 (COX-2), a prostaglandin G/H synthetase that is specifically increased in inflammatory processes (8,9). Chronic inflammation results in increased production of reactive oxygen species and a decrease in cell-mediated immunity, two factors that appear to influence the progression of viral infections, such as hepatitis B and C, to cancer, since these infections do not cause cancer unless chronic inflammation occurs (10). An expanding body of literature suggests that cervical carcinogenesis is also associated with inflammation. High rates of cervical cancer often coincide with endemic and epidemic cervicitis. A recent study (11) demonstrated an association of cervicitis with high-grade cervical lesions among oncogenic HPV-infected women. Another study (12) reported increased COX-2 expression in human cervical cancer, suggesting that inflammation is linked to cervical carcinogenesis. Of interest, a recent study (13) has shown that COX-2-mediated prostaglandin E2 (PGE2) increases interleukin 10, which inhibits dendritic cell production of interleukin 12, a critical cytokine in cell-mediated immune responses. Thus, chlamydial bacterial products may also promote HPV persistence through a functional decrease in antigen-presenting cells of the lower genital tract and inhibition of cell-mediated immunity. Importantly, a shift from T-helper lymphocyte type 1 responses (Th1) (cell-mediated immunity) to T-helper lymphocyte type 2 (Th2) (humoral immunity) is known to occur during chronic inflammation (10). It is well established that Th1 responses are important for host response to infectious diseases and other intracellular pathogens, and it is likely that Th1 responses are critical to the clearance of HPV infections (14). One study of in vitro stimulation of peripheral blood lymphocytes with HPV antigens (15) found the levels of in vitro interleukin 2, a marker for Th1 responses to HPV antigens, to be negatively associated with disease status. A large shift from Th1 to Th2 cytokine production, favoring humoral immunity over cell-mediated immunity, has also been associated with more extensive HPV infection (16). Few epidemiologic studies have examined local mucosal immune responses. Antioxidant Nutrient Status Over the past several decades, there have been numerous studies to examine the relationship of diet and nutrient status with cervical neoplasia and cancer risk. However, much of this work was conducted before a reliable measure of the central risk factor for the disease, HPV infection, was available. Few studies controlled for factors such as smoking and oral contraceptive use that are both related to cervical cancer and nutritional status. Moreover, most studies of cervical cancer were conducted before reliable high-pressure liquid chromatographic methods for separating and quantifying the major carotenoids and their geometric isomers in serum were available. In addition, the database for carotenoid content in the food supply has only recently been expanded to include carotenoids other than β-carotene (17). Finally, most studies used a retrospective design and, therefore, were unable to examine when during cervical carcinogenesis nutrients may have been active. Of six studies that properly controlled for HPV, inverse associations between serum β-carotene and cervical carcinogenesis have been observed when risks for CIN (18–20) and invasive cervical cancer (21,22) were examined. In studies that measured carotenoids other than β-carotene (21–25), significant inverse associations have been observed for serum lycopene and CIN (24,25) and serum α-carotene and invasive cervical cancer (24). Among the seven studies that assessed circulating tocopherol (vitamin E) concentrations (17,20–22,24,26,27), four found inverse associations with risk of CIN (20,24,26,27) and one with risk of invasive cervical cancer (17). Although diet has been associated with reduced risk of CIN and cervical cancer in several observational studies, the results from experimental studies have been negative (28). Eight phase III nutrient-based chemoprevention trials have been completed: two folic acid trials, five β-carotene trials, and one topical retinoic acid trial. Among these trials, significant improvement of cervical lesions was observed only in the retinoic acid trial (29). However, the failure of phase III clinical trials to show a treatment effect may be the result of not testing the nutrient at the point in carcinogenesis where it is most effective, treating for too short a period of time with single nutrients, and using pharmacologic, rather than dietary, concentrations of the nutrient. In support of the hypothesis that antioxidant nutrients may only be active early in cervical carcinogenesis is the observation that dietary lutein and vitamin E and circulating concentrations of lycopene were inversely associated with the reduced risk of oncogenic HPV persistence among young U.S. women (30), and lower serum β-carotene, β-cryptoxanthin, and lutein concentrations were associated with HPV persistence among U.S. Hispanics (31). Similar to these findings, dietary consumption of β-cryptoxantin, lutein, and vitamin C was inversely associated with persistent type-specific HPV infection among Brazilian women participating in the Ludwig–McGill HPV Natural History Study (Giuliano AR, Siegel EM, Roe D, Ferreira S, Baggio NL, Galan L, et al.: unpublished data). Together, these data suggest that certain carotenoids, such as lutein, may influence the natural history of HPV infections; however, another completed study (32) found no association with circulating concentrations of retinol, α- and β-carotene, or lycopene. It is important to note that all of these studies used relatively short times of persistence (6 months for the U.S. women and 12 months for the Brazilian women). Another concern is that different studies have attributed the protective effects of antioxidants to different micronutrients. These differences will need to be adjudicated. Future studies are needed to examine these associations in other populations and to evaluate whether markers of antioxidant nutrient status are influencing HPV persistence as defined by longer time periods (>1 year) and are associated with a decreasing oxidant load. Biologic Plausibility Factors that increase reactive oxygen species, such as smoking, inflammation, and reduced antioxidant activity, may adversely affect the natural history of HPV infections by increasing the oxidant : antioxidant balance and by directly influencing HPV transcriptional activity. As such, the cofactors discussed here and others presented may share a common mechanism. The association between oxidant load and carcinogenesis has been well studied. Early research indicated that reactive oxygen species oxidize cellular proteins and DNA that could lead to lethal mutations and a decrease in the host immune system. More recent research indicates that the role of reactive oxygen species is much greater than just one of damaging cellular proteins and DNA. Reactive oxygen species appear to have a central role in cell signaling by activating activator protein-1 and nuclear factor kappa B (transcription factors), cell proliferation, and apoptosis (33). These findings are particularly relevant to cervical carcinogenesis where viral replication (viral load), transcriptional activity (expression of HPV type 16 [HPV16] E6 and E7 proteins), cell proliferation, and apoptosis are pivotal events in cervical carcinogenesis. Examples from human immunodeficiency virus (34) and influenza virus (35) indicate a role for antioxidants, in particular, nutrient antioxidants, in the decrease of viral replication and expression. Evidence is accumulating to suggest that reactive oxygen species and their neutralization by antioxidants may work in a similar manner in HPV infection. The addition of antioxidants to in vitro assays has been shown to inhibit activation of the transcriptional factor AP-1, a central transcription factor for the expression of the E6 and E7 proteins from the oncogenic HPV types (36,37). Using an HPV16-immortalized human keratinocyte culture, Rosl et al. (38) demonstrated that the antioxidant pyrrolidine dithiocarbamate selectively suppressed AP-1-induced HPV16 gene expression. These investigators suggested that manipulation of the reduction-oxidation (redox) state may be a novel therapeutic approach to interfere with the expression of oncogenic HPV. To further examine the role of reactive oxygen species in cervical neoplasia, new methods to sample the cervical microenvironment are needed, as are reliable biomarkers of oxidant load and its effects on HPV. If cervical inflammation promotes the progression of HPV infections to cervical precancer and cancer, we predict the following confirmatory evidence: First, COX-2 or other inflammatory biomarkers will be increased in persistently HPV-infected cells before the development of precancer and cancer. Second, inhibitors of inflammatory pathways (e.g., Celecoxib, a COX-2 inhibitor) will be protective against the development of cervical precancer and cancer but will be unlikely to reverse cervical precancer and cancer. Finally, either higher content of dietary antioxidants will protect against and/or deficiencies will be a risk factor for cervical precancer and cancer. New Technologies for Measuring the Cervical Microenvironment A complete understanding of HPV cofactors will require the use of new technologies that can directly assess changes to the local cervical microenvironment in response to HPV exposure and through progression to CIN3 and invasive cervical cancer. Several new cervical sampling and testing methods may prove to be useful in future studies of HPV cofactors. Cervical Secretion Sampling Whether exposure to HPV cofactors is systemic (e.g., smoking or oral contraceptives), local (e.g., STIs), or both (e.g., multiparity, which may act through a hormone-related mechanism or cervical tissue damage), each must act directly on the cervical epithelium to exert its biologic effect. Although HPV-infected cervical cells may be ideal for examining the biologically effective exposure dose and its biologic consequences, cervical secretions may be useful biospecimens for the measurement of the cervical microenvironment. Cervical secretions can be used to measure environmental exposures, as has been shown for measuring smoking metabolites in smokers. Although these measurements are no more accurate than the assessment of smoking by questionnaire, the presence of the smoking metabolites in secretions is evidence of a biologic role of smoking in cervical carcinogenesis. Cervical secretions can also be used for the measurements of local immunity (and inflammation), which is likely to be more accurate for the assessment of immune response to the HPV than systemic measurements, since immunity at the cervix is composed of locally derived immunity via the mucosal immune system and blood-borne immune factors that arrive at the cervix via transudation. For example, blood concentrations of interleukin 10 and interleukin 12, important for the regulation of Th1 and Th2 immune responses, were not correlated with levels in cervical secretions (39). However, methodologic issues have hindered multiple measurements from cervical secretions. First, it has been difficult to collect cervical secretions without perturbation of the cervical epithelium and the influx of serum proteins and cells or contamination with vaginal secretions. New methods, such as using absorbent ophthalmic sponges placed at the os of the cervix (40), have proven to be useful in overcoming these difficulties. Cervical secretion specimens are typically approximately 50 μL and, therefore, there is a limited number of measurements that can be made per specimen. Thus, despite the relative ease of collecting cervical secretions as part of the Pap screening visit, their collection has only been implemented recently in large cohort studies. Similarly, it may be necessary to measure local micronutrients to determine actual exposure. Although α- and β-carotene and folate were highly correlated in cervical secretions and cervical tissue and plasma (41–43), other micronutrients did not appear to be correlated (42). When blood and cervical tissue and secretion micronutrient levels are poorly correlated, local measurements are necessary. However, current micronutrient assays have large tissue or specimen requirements, making the use of cervical specimens for nutritional analyses limited. Moreover, many micronutrients are lipid soluble and thus can only be measured from tissue. Improving the assay sensitivity of tissue-based or secretion-based micronutrient assays is needed to permit their use in epidemiologic studies. However, we emphasize that we have begun to pilot measurements of cervical secretions, and there are a number of important considerations that will need to be addressed to assess the overall usefulness of these specimens. First, we do not understand the relationship of the measurements in secretions to the biology of the cervical tissue. Second, it will be necessary to demonstrate that these measurements are sufficiently reproducible and reliable that they provide a more accurate measure of immunity than measurements of plasma or serum. Third, there is uncertainty about what these measurements represent in terms of the duration and intensity of the targeted effect (e.g., exposure, damage, or immune response). Methodologic studies, such as correlation studies that examine the relationships between secretions and tissue and between multiple collections over time, are necessary to validate the use of measurements in epidemiologic studies. New Assays To compensate for low sample volume, new “multiplex” technologies can be applied to efficiently detect multiple biomarkers (e.g., cytokine profiles) from these small-volume (e.g., <50 μL) specimens. These include recycling immunoaffinity chromatography (RIC) (44), flow cytometry-based systems (45), and protein microarrays (46). In pilot studies using RIC, we have successfully measured greater than 20 immune markers from an equivalent of 3 μL of a cervical specimen with excellent reproducibility on split aliquots (Spearman correlations and kappa values exceeding 0.88 and 0.78, respectively, for all immune markers) (Castle PE: unpublished data). A main requirement for these technologies is the availability of antibodies or other capture molecules against target biomarkers. Thus, these technologies are not limited to immune markers and may be used to measure other biomarkers with the appropriate capture molecules available. For example, in the same pilot study using RIC, we have been able to measure COX-2 in cervical secretions. If the appropriate antibodies were available, it is theoretically possible to measure different DNA adducts from lysates of cervical cell specimens. Biomarker Measurements From Cervical Pap Specimens Alternatively, Pap specimens might be used for detection of biomarker or measures of the local microenvironment. One pilot study (47) demonstrated that cell-mediated immunity could be measured in vitro from cervical Pap specimens challenged with HPV antigens (cervical cell-mediated immunity). Another study (48) demonstrated the feasibility of collecting Pap specimens into 4 M guanidine thiocyanate-based buffering solution and performing reverse transcription–polymerase chain reaction (RT–PCR) for the measurement of cytokine expression. As with measurements from cervical secretion, the reproducibility, reliability, and validity of these measurements must precede their application to epidemiologic studies. The limitation of these approaches may be the applicability to large epidemiologic studies. For example, cervical cell-mediated immunity likely must be conducted in real time, which limits the number of specimens that can be handled and presents logistical problems for remote field efforts. Therefore, cervical CMI may only be applicable to smaller follow-up studies of women known to have had a type-specific oncogenic HPV infection, with outcomes of viral clearance, viral persistence without disease, and progression to CIN2 or more severe diagnoses (CIN2 is the threshold diagnosis for treatment) assessed. Standard antigens, such as recall antigens (e.g., influenza) and mitogens, may help to standardize these assays across different studies. For RNA-based measures of cervical tissue, denaturation for RT–PCR may limit its use for other biomarker measures. Furthermore, methodologic development (e.g., cytologic media that are compatible with HPV DNA testing methods and preserve messenger RNA for RT–PCR) will permit the translation of these assays from pilot studies into broad usage in epidemiologic studies. Final Comments One important tenet of causality is temporality. Given the general limitations of case–control studies for causal inference in combination with those statistical quandaries that are particular to studies of HPV (49), it does not appear necessary to proceed with more case–control studies to examine the role of certain cofactors, such as C. trachomatis and HSV-2, in the development of high-grade cervical neoplasia. The case–control design does not yield data that inform whether these STIs are surrogate markers for high-risk behavior and increased number of HPV infections or markers of repeated exposure to semen, which is known to contain high concentrations of inflammatory mediators such as PGE2 that may contribute to cervicitis. Cervicitis could also obscure cytologic detection of problematic lesions, thereby increasing the risk of the development of more severe neoplasia before detection. Thus, carefully constructed prospective studies are needed to evaluate the roles of STIs, cervical inflammation, and antioxidants (oxidant-to-antioxidant ratio) in the natural history of HPV infection. Prospective studies of HPV-infected women, with outcomes of type-specific HPV persistence and progression to precancerous lesions (instead of cancer) versus viral clearance, might be more efficient than standard designs. Such studies could be initiated from Pap screening programs in which women with Pap smears interpreted as low-grade squamous intraepithelial lesions (LSILs) could be recruited. A high percentage of these women (≈80%) will have HPV, and many (≈30%) will have an oncogenic type and will not have undergone a censoring treatment. For studies aimed more at evaluating progression to cervical precancer and cancer given persistent HPV infections, women with consecutive yearly abnormal Pap smears (atypical squamous cells or LSILs) can be recruited, accepting that there will be some misclassification of type-specific persistence. Within prospective studies, the STIs, concurrent with HPV infection, can be assessed using PCR, ligase chain reaction, and Hybrid Capture DNA assays instead of inferred concurrent STI exposure using serologic measures in case–control studies. Multiple biomarker measures will permit an examination of the mechanisms (viral load, viral persistence, and genotoxicity) by which potential HPV cofactors exert their effect. Case–control studies will be useful in piloting and evaluating new techniques to measure biomarkers at the cervix. One of the critical issues will be what cervical specimens, cervical secretions (to be used for protein measurements), or cervical cells (for RNA measurements) should be collected for these measurements. The relationship of these two measurements is largely unknown, although a recent pilot study (50) found that the cytokine levels in the fluid phase of cervicovaginal lavage did not correlate with RT–PCR levels from cellular material in the lavage. Methodologic issues, such as specimen collection and storage and development of sensitive, specimen-efficient (multiplex) assays, will be critical to the success of epidemiologic studies attempting to examine the cervical microenvironment in relation to the natural history of HPV infection. Reproducibility of cervical sampling is absolutely critical to the success of using these measurements in the context of HPV natural history studies. Another important tenet of causality is biologic plausibility. Indeed, the evidence for HPV, including biologic plausibility, as the causal factor for cervical cancer is overwhelmingly strong. Epidemiologic studies have identified a set of additional risk factors, HPV cofactors, which may influence the natural history of an HPV infection. Just as measuring HPV DNA at the cervix has become a required component of etiologic studies of cervical cancer, measurements at the cervix will be necessary to link epidemiologic evidence of a purported HPV cofactor to its biologic effects. New methodologies and technologies may facilitate such measurements, but like the development of accurate HPV assessment that required years of metholodogic work before it was sufficiently reliable, reproducible, and valid, measurements of the cervical microenvironment are at their infancy and may require similarly rigorous efforts. Without such efforts, epidemiologists will be awash with data but left with the uncertainty of whether the relationship between an HPV cofactor and the risk of neoplasia is real or artifact as the result of residual confounding by HPV, which can only be partially overcome by statistical methods. 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JNCI Monographs – Oxford University Press
Published: Jun 1, 2003
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