Risks for Incident Human Papillomavirus Infection and Low-Grade Squamous Intraepithelial Lesion Development in Young Females FREE

Low-grade squamous intraepithelial lesions (LSILs) are relatively common manifestations of cervical human papillomavirus (HPV) infections. Most pathologists agree that LSIL is a cellular response to HPV infection^1- 2 resulting from the expression of specific viral proteins that interact with the host cytoskeletal structures and induce cell proliferation.^3- 5 Some researchers suggest that all HPV infections result in pathologic changes of LSIL at some point in the natural history of infection; however, the discrepancy between rates of infection with HPV and development of LSIL is quite striking, even in adolescent and young women in whom the prevalence of both is high. Rates of HPV infection in sexually active young females have been consistently reported to range from 19% to 46%; these are significantly higher than reported rates of LSIL, which range from 1.1% to 7.0% in the same groups.^6- 8 Differences in the length of HPV and LSIL persistence do not completely explain the observed discordance because recent studies have reported that most females who have HPV infection clear it within 12 to 24 months,^9- 10 a rate that is similar to the regression rate for LSIL in young women.^11- 12 Consistent discrepancies suggest that the development of LSIL is a distinct event in the natural history of HPV infection. Consequently, risks associated with LSIL should be distinct from risks for infection with HPV.

While studies have shown that risks for HPV and LSIL, specifically those associated with sexual behavior, significantly overlap,^13- 14 many studies have found that certain behavioral and biological risks that seem to be relevant to the development of LSIL are not significant risks for infection with HPV,^6,15- 18 suggesting that HPV alone is not sufficient for the development of LSIL. From an epidemiological perspective, behavioral and biological risks for LSIL are difficult to distinguish from risks for HPV infection in cross-sectional studies because all females with LSIL have HPV, and females without LSIL are less likely to have HPV infections.

The purpose of the current investigation was to determine the risk factors for incident cervical LSIL development in a cohort of adolescent and young women with HPV infections, and the risk factors associated with acquiring an HPV infection for females who tested negative for HPV DNA at entry into the study.

The females in this report were part of an active, ongoing natural history study of HPV infection that began in 1990.^6,10 Females gave consent according to the guidelines set forth and approved by the Committee for Human Research, University of California, San Francisco. We screened females aged 13 to 20 years who were attending 1 of 2 family planning clinics between 1990 and 1994 for detection of cervical HPV DNA using a commercial test (HPV Profile, Digene Diagnostics, Silver Spring, Md). We asked those who tested positive to participate in the study. The characterization of this cohort recruited using the commercial test is described in detail elsewhere.^6,10 We replaced testing for HPV DNA by the polymerase chain reaction (PCR) technique in 1995. In addition, we retested all samples retroactive to baseline using PCR. All data in this article refer to HPV DNA using PCR amplification.

For the HPV incidence study we included only females who entered the study negative for HPV using PCR. Because of the possible false negatives, which have been associated with HPV testing,¹⁹ we used conservative criteria for defining initial HPV negative tests: only those from the cohort who at baseline and first follow-up had negative results for HPV using PCR were included for the incident HPV study. For the LSIL incidence study we included only females who entered the study negative for LSIL. Because of the known false-negative rate (approximately 50%-70%) of cytology for squamous intraepithelial lesions, we defined similar conservative criteria for the LSIL incidence study by including only females from the cohort who at baseline and first follow-up had normal cytologic findings. In addition, individuals in the HPV incidence study who developed an incident HPV infection were included in the LSIL analysis. However, we excluded from the LSIL incidence study those who did not test positive for HPV by PCR at any time during the study, because most scientists agree that HPV is necessary for LSIL development.² Including HPV-negative subjects in the LSIL analysis may have diluted the risk factor analysis. The median number of visits was 9 (interquartile range [IQR], 4-15) and the median time in the study was 50 months (IQR, 23-79 months) for females who were followed up under the above criteria.

Visit protocols are detailed elsewhere.¹⁰ Baseline and interval visits on all HPV-positive (every 4 months) and HPV-negative (every 6 months until they turned HPV-positive, and then every 4 months) participants included cervical cytology, testing of cervical samples for HPV DNA collected using sterile Dacron swabs, and face-to-face interviews to obtain information on demographics and sexual and substance use behaviors. Colposcopic examination of the vulva, vagina, cervix, and perianal areas was also performed on all subjects with the aid of 3% acetic acid at each visit. Cervical samples were obtained for Chlamydia trachomatis and Neisseria gonorrhoeae, and vaginal samples were collected, measured for pH, and examined microscopically for the presence of clue cells and Trichomonas vaginalis. These latter cervical and vaginal samples were taken at the baseline and annual examinations, as well as at interval visits for those females who were currently symptomatic for lower genital tract infections. C trachomatis was identified initially using standard culture techniques.²⁰ In 1996, we replaced the culture test by the ligase chain reaction technique (LCX STD system, Abbott Laboratories, Abbott Park, Ill), which did not significantly alter the incidence of C trachomatis (annual incidence of <3% for each). For purposes of this study, bacterial vaginosis was diagnosed if 2 of the following 3 criteria were met: vaginal pH greater than 5.0, 20% or greater of cells on wet mount clue cells, and a positive "whiff" test using potassium hydroxide.²¹ Herpes serology²² using Western blot was performed on the majority of subjects at baseline. During follow-up, viral cultures were performed only when signs or symptoms (ulcers or severe cervicitis) were present at any given visit. We diagnosed herpes simplex infections as follows: if a subject tested positive for serology at baseline or at any other visit prior to LSIL diagnosis; if a laboratory test for herpes simplex virus (HSV) yielded a positive result; or if a subject reported a history of HSV at baseline or between visits.

At baseline, all females with areas of atypia suggestive of high-grade squamous intraepithelial lesions (HSILs) as defined on colposcopy²³ were randomly assigned to biopsy or no biopsy. A total of 53 females received biopsy per this protocol. In addition, females with lesions consistent with carcinoma in situ by colposcopy²³ (n = 15) also underwent biopsy. This method allowed for a more accurate diagnosis and characterization of lesions present at entry. A subset of this group and results of histology based on this protocol were published earlier.²⁴ An analysis of the potential effect of these biopsies on the natural history of HPV was conducted and no effects were found.

We tested cervical samples at baseline for HPV DNA using HPV Profile (Digene Diagnostics, Silver Spring, Md). All samples have been subsequently retested using 1 of 2 PCR techniques. Methods for the initial amplification technique have been described previously.¹⁰ Our initial method sought the presence of amplified HPV material in a dot blot format using an enhanced chemiluminescent method. A generic probe mix was used that determined the presence of 1 or more of 25 different HPV types nonspecifically and β-globin for internal consistency. Samples were also specifically probed for low-risk HPV types 6/11/42/44, and high-risk HPV types 16, 18, 31/33/35, 39, 45, 51, 52, 56, and 58.

Our second method for amplification and detection used Roche reverse blot "strip" method (Roche Molecular Systems, Inc, Alameda, Calif).²⁵ A denatured biotin-labeled PCR product was hybridized to an array of immobilized oligonucleotides: HPV types 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 55, 56, 58, 59, 68, MM4 (W13B), MM7 (P291), MM9 (P238A), 6, 11, 40, 42, 53, 54, 57, 66, MM8 (P155), and 2 β-globin controls. Of the 7114 HPV samples tested, we tested 1957 using the strip method and 5157 using the dot blot test. A total of 156 were tested by both methods. We found agreement between the 2 tests to be 87.8%, yielding a κ statistic of 0.63, which indicates substantial agreement statistically (P<.001).

Since we did not use the strip method for all samples, the type-specific data used for this analysis included only HPV types for which all subjects were screened at all visits. We classified all other positive HPV types by the strip method as HPV type "other" and HPV generic probe positives by dot blot with no type identified were also classified as HPV type "other."

When the study began in 1990, we used standard World Health Organization cytologic nomenclature for condyloma and cervical intraepithelial neoplasia (CIN) grades 1-3. We asked the females who were diagnosed with CIN 1-3 by cytology at any given visit to return for biopsy. For purposes of this analysis, we categorized diagnoses per the Bethesda system for rating cytology using the nomenclature LSIL and HSIL.²⁶ We diagnosed LSIL cases in this study based on cytology; HSIL cases were classified based on a diagnosis of HSIL on either cytology or histology.

We first made estimates of the distribution of time to HPV-positive results for females screened as HPV-negative by PCR at entry using flexible parametric regression methods for interval-censored survival outcomes.²⁷ The hazard function of this distribution was modeled as a polynomial (spline) function of duration of follow-up and likelihood ratio tests were used to select the best-fitting model among fitted alternatives. We made estimates of the distribution of time to initial diagnosis of LSIL from the time of initial HPV infection using a generalization of the method described above to account for additional uncertainty about the time of initial HPV infection. For the females observed to be HPV-positive at entry, initial infection is known only to fall in the interval between initiation of sexual activity and enrollment. For the females screened as initially HPV-negative, we assumed the time of initial HPV infection to be uniformly distributed in the interval between the last observed negative and first positive test results.

We used a discrete-time version of the proportional hazards model allowing for interval censored outcomes and incorporated time-dependent covariates to examine risks for the development of both LSIL for females with preceding positive results for either high- or low-risk HPV types, and for the first incidence of HPV for those females who entered the study with HPV-negative status.

In the incident LSIL analysis, we treated variables pertaining to HPV infection in several ways. The risk associated with current HPV status at each visit (including high-risk, low-risk, and any HPV type) was examined. In addition, we examined the risk related to length (months) of persistent infection with the same HPV type (ie, the same type within each individual; types were allowed to vary among individuals). We also subdivided this latter variable into categories of positive (with the same type) for less than 1 year, 1 to 2 years, 2 to 3 years, and more than 3 years. We then entered these categorical variables into the analysis, using HPV negativity as the baseline to which each duration category was compared.

We first examined each variable in a univariate analysis and, using a significance level of .05, entered those variables found to be significant into a multivariable analysis for each of the 2 outcomes, incident HPV infection and diagnosis of LSIL. Variables that lost significance at the .05 level in the multivariable analysis were excluded from the final models. P values were calculated using the Wilcoxon nonparametric test for continuous variables and the χ² test for categorical variables. Statistical software used was SAS version 6.12 (SAS Instititute, Cary, NC) and S-PLUS version 5.0 (Insightful Corp, Seattle, Wash).

Of the 897 females who consented to participate in the study, 81 females had incomplete data at baseline (missing questionnaires, missing Papanicolaou test data, or inadequate PCR samples) and were not included in the study. A total of 155 had a prevalent LSIL at entry (baseline or first follow-up visit) and were excluded. In addition, we excluded 17 females who developed HSIL prior to LSIL. Of the remaining 644 females, 105 were HPV-negative using PCR at entry (baseline and first follow-up visit) and eligible for the HPV incidence study; 51 of these never developed an HPV infection and were excluded from the LSIL analysis. In addition, 97 females had fewer than 3 visits and were considered lost to follow-up for the LSIL analysis, resulting in a total of 496 females for the LSIL analysis.

Analysis comparing the latter females lost to follow-up (n = 97) with those included in the LSIL study revealed no significant differences in terms of race, age at entry into the study, years sexually active, number of recent partners, or sexually transmitted disease (STD) history. However, these females had significantly fewer sexual partners at baseline (median, 4; IQR, 2-7 vs median, 5; IQR, 3-9 for those in the study, P = .03) and were of lower socioeconomic status, as measured by the education levels of their mothers (40.7% had completed some college education in the excluded group vs 53.9% in the analysis group, P = .02).

Demographic characteristics of and comparisons between the 2 groups (HPV and LSIL incidence cohorts) are given in Table 1. The group HPV-negative at entry had fewer lifetime sexual partners, but recent sexual behavior as defined by the number of partners reported in the last 2 months was not significantly different for the 2 groups. No other significant differences between the 2 groups were found.

There were 54 incident cases of HPV infection. Forty-six percent of the HPV infections were with high-risk types only, 9% were with low-risk HPV types only, 13% were with mixed high- and low-risk HPV types, and 32% were with a type other than those in the standard mix. Figure 1 shows the estimated distribution of time for subjects remaining free of HPV infection. The estimated proportions of females infected at 12 and 36 months following enrollment were 0.17 (95% confidence interval [CI], 0.14-0.19) and 0.55 (95% CI, 0.31-0.79), respectively. (These estimates are obtained from the corresponding estimates and CIs for the probability of remaining uninfected in Figure 1 by subtraction from 1.) The curve predominantly reflects high-risk HPV types since the majority of the group had high-risk infection. The median time of follow-up for the females remaining HPV-free was 26 months (IQR, 10-54 months).

The univariate analysis of risks for cervical HPV infection adjusted for time in study is shown in Table 2. Multivariable analysis is summarized in Table 3. We found significant risks for infection with HPV to be sexual behavior, history of HSV, and a history of vulvar warts. The current use of oral contraceptives when included in the multivariable analysis continued to have a significantly protective effect. Two variables that had been significant in univariate analysis, history of marijuana use and lifetime sex partners, lost significance in the multivariable analysis and were not included in the final model.

Ten (19%) of the 54 females with incident HPV developed LSIL. Among them, 7 had a high-risk HPV type, 1 had a low-risk type, and the remaining 2 had unknown HPV types. There was no difference between the proportion of those with incident high-risk types who developed LSIL and those with incident low-risk HPV types. One participant tested positive for HPV after the LSIL diagnosis. No difference was found between those positive for low- and high-risk types for time from HPV infection to LSIL diagnosis.

The HPV type distribution at baseline or first incidence in the LSIL study (n = 496) was as follows: 75 were positive for types 6/11/42/44; 134 for type 16; 62 for type 18; 57 for types 31/33/35; 23 for type 39; 27 for type 45; 44 for type 51; 54 for type 52; 15 for type 56; 32 for type 58; and 282 were positive for other HPV types. These numbers do not sum to the sample size of 496 because some individuals were positive for multiple types: 96 for 2 types and 81 for 3 or more types.

There were 109 incident cases of LSIL. Figure 2 shows the estimated distribution of time for subjects remaining free of LSIL. The estimated proportions of individuals developing LSIL at 36 and 60 months following initial HPV infection were 0.15 (95% CI, 0.13-0.17) and 0.21 (95% CI, 0.17-0.25), respectively. After 60 months, the estimated proportion developing LSIL remained relatively flat, reflecting few observed infections among females with longer follow-up. The median time of follow-up after HPV detection for those remaining LSIL-free was 60 months (IQR, 28-82 months). No differences assessed by overlapping pointwise 95% CIs between high- and low-risk HPV types were found for time to LSIL (curves not shown).

Table 2 shows the univariate analyses of risk factors for LSIL adjusted for time in study. Those females who were HPV-positive at the time of visit were approximately 7 times more likely to develop LSIL than those who were negative. The relative hazard for current infection with a high-risk HPV was 5.39 (95% CI, 3.57-8.15) vs a relative hazard for current infection with a low-risk type of 10.68 (95% CI, 4.80-23.78); however, this latter estimate was far less precise, because infections with low-risk types alone made up only 4% of the total positive HPV tests and were, therefore, infrequent vs infection with high-risk types (63% were positive for only high-risk types, 2% were simultaneously positive for both high- and low-risk types, and 31% were positive for HPV type "other").

In order to examine the association between HPV persistence and the development of incident LSIL, we also examined the duration of type-specific HPV infections as a risk factor for LSIL. We found that each additional year of HPV type-specific positivity increased the relative hazard by slightly more than 2-fold (2.27; 95% CI, 1.86-2.78). To investigate whether this relationship was strictly linear over time, a quadratic term in time was added to the model and was found to be significant at the .07 level. We also examined a categorized version of our duration variable, in which HPV persistence was divided into intervals of positive for less than 1 year, 1 to 2 years, 2 to 3 years, and more than 3 years. While there was some indication of a leveling off of risk after 2 years of infection, the small size of the last category prevented us from reaching any firm conclusions regarding nonlinearity.

All factors that were significant in the univariate analysis at the .05 level were included in a multivariable analysis. We used the categorized HPV infection duration variables rather than those indicating high- and low-risk status at visit to characterize HPV infection in the multivariable analysis, because so few subjects were positive for low-risk HPV types at the time of LSIL diagnosis. Therefore, estimates for the categorized variables were more precise. Presence of HPV and cigarette smoking remained significant. Marijuana use and number of lifetime partners lost significance in this analysis and were not included in the final model (Table 4). Interaction terms were tested in the model, but were excluded from the final model since they were not significant.

Although LSIL is considered a common benign manifestation of HPV infection, the findings from this study underscore the differences in the biological and behavioral risks associated with the acquisition of HPV as compared with the development of LSIL. Our results contrast with many cross-sectional studies.^13- 14,28 Since HPV infection and LSIL are strongly associated, incident studies are better epidemiological tools to address differences in risk. Not surprisingly, a current positive HPV test result with either low- or high-risk HPV types was the strongest risk factor for LSIL. Sexual behavior, specifically exposure to new partners, represented the strongest risk factor for incident HPV infection.

The association between sexual behavior and incident HPV is quite notable if the actual risk is taken into account. That is, risk increased nearly 10-fold for each new partner per month reported. The strong association between sexual behavior and HPV infection supports the hypothesis that HPV is a sexually transmitted virus and that most "new" infections in young females are predominantly due to exposure, rather than the "transient" up-regulation of latent infections in nonimmunocompromised hosts.

The association with vulvar warts is not surprising because the virus can easily be transmitted from the vulvar area to the cervix via intercourse or from other sources, including tampons. The association between incident HPV and the history of HSV is most interesting because HSV has frequently been associated with the development of cervical cancer.²⁹ It is well known that primary and secondary HSV infections result in breaks of the cervical mucosal barrier through inflammation and ulceration, thereby granting easy access to the basal epithelial cells for viruses such as HPV. The lack of association with other risk factors supports the strong role of sexual exposure in HPV acquisition and diminishes support for other behaviors that most likely reflect risky sexual behavior of the female or her partner.

The independent protective role of oral contraceptives in acquiring HPV is also interesting since prolonged use of oral contraceptives has been associated with development of squamous intraepithelial lesions in several studies.^16,30 Since condom use was not significant in the univariate analysis, we postulate that this relationship may be due to the influence of estrogen or progesterone on immune regulation.³¹

Lack of association between sexual behavior and LSIL also underscores the fact that in addition to HPV, other risks are necessary for LSIL development. Indeed, in this study we found that daily cigarette smoking had a deleterious effect, which contributed to the development of LSIL. The mechanism for this is not understood; however, several epidemiologic studies have found a role for cigarette smoking in invasive cervical cancer.³² Nicotine and other cigarette metabolites have been found in the cervical mucus.³³ We suspect that the relationship of cigarette smoking to LSIL may result from its ability to cause immune dysregulation.³⁴ As discussed, LSIL is a histological result of active HPV replication that may involve up-regulated protein transcription. Several studies have implicated the immune system in viral regulation.^35- 39

The lack of association between LSIL and other risk factors such as contraceptive use or C trachomatis infection as found in other studies^13,40 may be due to several factors. First, the cohort falls into quite a young age group, where the squamous intraepithelial lesion is predominantly LSIL. That is, many of the other studies using older populations have defined LSIL by cytology alone, potentially resulting in the underdiagnosis of HSIL.⁴¹ Consequently, these studies have identified risk factors for a group with mixed HSIL and LSIL. The majority of our participants with LSIL underwent biopsy to rule out HSIL. Second, HPV infection is a required risk for LSIL. Our population appears to frequently contract new HPV infections, whereas in older females with less frequent rates of sexual activity, HPV DNA detection more often reflects a persistent infection.⁹ Third, risk factors such as oral contraceptive use may require years of exposure before the "risk effect" is seen. Again, our participants were relatively young with less than 4 years of oral contraceptive exposure. The lack of association between STD infections and LSIL may have been due to the low prevalence of STDs in this population.¹³ However, we emphasize again that STDs may be a measure of risk behavior. Risk behavior connotes HPV exposure, which in turn increases the risk of the occurrence of LSIL.

Although LSIL was strongly associated with HPV, the majority of young females in our study with HPV infection never developed LSIL, suggesting that certain biological risks or conditions are necessary in order for the LSIL to occur. In addition, the interval between HPV infection and LSIL varied, implying that in some females a period of latency occurs before LSIL develops. The reason for this latency and later up-regulation is not clear. Our findings suggest that the risk of developing LSIL in young females is only present within the first 3 years after detection of HPV DNA. There was no evidence that the risk continued after 3 years; however, the analysis was limited due to small numbers in this group. Since this study was performed on young females, the risk cannot be generalized to older women.

In summary, there were clear differences in risks for incident HPV infection and LSIL development, suggesting that many of the risks that have been associated with LSIL in cross-sectional studies were either risk factors for or surrogate markers of HPV infection. The strongest risk for HPV detection in young female is sexual exposure. However, the majority of females in this study with a positive HPV result never developed LSIL within a median follow-up time of 60 months, which suggests that certain biological risks or conditions are required for the LSIL to occur. One such risk in our group was the use of tobacco, a known carcinogen.