Results A total of 587 vaccine and 763 placebo recipients underwent cervical surgery. The incidence of any subsequent HPV related disease was 6.6 and 12.2 in vaccine and placebo recipients respectively (46.2% reduction (95% confidence interval 22.5% to 63.2%) with vaccination). Vaccination was associated with a significant reduction in risk of any subsequent high grade disease of the cervix by 64.9% (20.1% to 86.3%). A total of 229 vaccine recipients and 475 placebo recipients were diagnosed with genital warts, vulvar intraepithelial neoplasia, or vaginal intraepithelial neoplasia, and the incidence of any subsequent HPV related disease was 20.1 and 31.0 in vaccine and placebo recipients respectively (35.2% reduction (13.8% to 51.8%)).

Current data show that HPV vaccination does not reduce progression to cervical precancers in women with ongoing infections at the time of vaccination. 5 10 11 However, no studies to date have looked at the impact of HPV vaccination in preventing subsequent disease after treatment for such precancers. In two recent phase III clinical trials of the quadrivalent HPV vaccine, more than 17 000 women were followed for approximately four years. Participants who underwent treatment for cervical, vulvar, or vaginal disease continued to be followed until the end of the study. We therefore had a unique opportunity to evaluate the post-treatment effect of vaccination in these women. In this retrospective analysis we identified women who had an excisional procedure for cervical disease or who were diagnosed with genital warts, vulvar intraepithelial neoplasia, or vaginal intraepithelial neoplasia after randomisation to vaccine or placebo and followed these women for additional disease outcomes. The objective of this study was to determine if the vaccine decreased the risk of developing subsequent disease after the first definitive treatment.

The currently available human papillomavirus (HPV) vaccines that are based on recombinant virus-like particles are designed to prevent HPV associated disease. Both the quadrivalent vaccine (against HPV types 6, 11, 16, and 18) and the bivalent vaccine (against types 16 and 18) are highly effective in preventing cervical intraepithelial neoplasia grade II-III or adenocarcinoma in situ in women who are not infected with the relevant HPV type before vaccination. 1 2 3 4 5 The quadrivalent vaccine has also been shown to prevent HPV related vulvar intraepithelial neoplasia grade II-III, vaginal intraepithelial neoplasia grade II-III, and genital warts in women and to prevent genital warts and high grade anal disease in men. 1 6 7 8 9

Kaplan-Meier estimates (and 95% confidence intervals) of cumulative incidence rates for subsequent disease were plotted with case counting starting 60 days after cervical surgery or vulvar or vaginal disease diagnosis. 18 The plots were not part of a formal survival analysis; rather they give a visual demonstration of the divergence of the incidence rates between the two vaccination arms over time. The 95% confidence interval for the Kaplan-Meier incidence at intervals of six months are not directly comparable with vaccine efficacy estimates. Vaccine efficacy was calculated from the exact conditional procedure and is included in the Kaplan-Meier plots as a reference.

Women contributed to person years at risk from 60 days after the initial surgery or diagnosis until the day of ascertainment of the subsequent disease end point, or, for those without a subsequent disease end point, until the day of their last follow-up visit. Incidence, or rate of subsequent disease, is expressed as the number of women with an end point per 100 person years at risk. A point estimate of vaccine efficacy (that is, percentage reduction of the relative end point) and the 95% confidence interval were calculated on the basis of the observed case split between vaccine and placebo recipients, adjusted for the accrued person time in each group. The criterion for statistical significance (P<0.05) was equivalent to requiring that the lower bound of the confidence interval for vaccine efficacy exclude 0%. An exact conditional procedure was used to evaluate vaccine efficacy under the assumption that the numbers of cases in the vaccine and placebo groups were independent Poisson random variables. 17

In the analyses (presented in tables 2–4 ⇓ ⇓ ⇓ ) a woman is counted only once for each end point (that is, once in each row), but a woman may have developed more than one end point during the trial (that is, a women may appear in more than one row). For example, a woman who is diagnosed with a cervical intraepithelial grade 3 lesion that is positive to HPV types 16, 31, and 45 would be counted once for (1) any disease; (2) any cervical disease; (3) any cervical intraepithelial grade II or worse; (4) any cervical intraepithelial grade III or worse; (5) any disease related to vaccine HPV types; (6) cervical intraepithelial grade I or worse related to vaccine HPV types; (7) cervical intraepithelial grade II or worse related to vaccine HPV types; and (8) cervical intraepithelial grade III or worse related to vaccine HPV types.

All specimens from biopsies and excisional procedures were tested for 14 HPV types (6, 11, 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, and 59) using an assay based on the polymerase chain reaction. 14 15 16 All tissue specimens underwent histopathological review by a pathology panel, who were masked to the women’s vaccination group and HPV status. A woman was considered to have developed an end point related to vaccine HPV types (HPV 6, 11, 16, or 18) or an end point related to 10 non-vaccine types (HPV 31, 33, 35, 39, 45, 51, 52, 56, 58, or 59) if the respective HPV DNA were detected in the same lesion that was diagnosed by the pathology panel as cervical intraepithelial neoplasia, vulvar intraepithelial neoplasia, vaginal intraepithelial neoplasia, or genital warts. For analyses of all disease, irrespective of causal HPV type, a woman was considered to have developed an end point if she had a lesion diagnosed by the pathology panel without consideration of HPV status.

As stated previously, women were referred for colposcopy and cervical surgery in accordance with a triage algorithm. 1 2 In FUTURE I, the pre-specified primary end points included all HPV related anogenital disease, thus the study included stringent criteria for disease determination and follow-up, including more frequent examinations and screening and more aggressive colposcopy triage for suspected disease. In FUTURE II, the primary end point was cervical intraepithelial neoplasia grade II-III and adenocarcinoma in situ. Treatment of vulvar or vaginal disease in FUTURE II was based on local standards of care, and the investigators were not required to record the date or method of treatment. We therefore examined the average time from diagnosis to treatment of genital warts, vulvar intraepithelial neoplasia, and vaginal intraepithelial neoplasia within the FUTURE I study. That information was used to determine an appropriate time frame after which most women would have been treated, so that follow-up after that window would probably capture new, rather than residual, disease. In FUTURE I, the mean number of days between diagnoses and treatment of genital warts, vulvar intraepithelial neoplasia, and vaginal intraepithelial neoplasia was 28 days (interquartile range 0–42 days). As the trials were less than four years of total duration (mean of 3.6 years), and since 82% had treatment within 60 days of the pathology panel diagnosis, we choose this as the cut off to ensure there would be enough follow-up time to detect subsequent disease after treatment. We assumed a similar time window between treatment and diagnosis in FUTURE II, and used the same window (60 days after diagnosis) for both studies. For consistency, the same time window was applied for counting subsequent disease after cervical surgery. A sensitivity analysis was also performed using a 90 day time window, as 91% of subjects had treatment within 90 days.

In this retrospective, intention to treat analysis, we identified all women who underwent definitive cervical therapy or who were diagnosed with vulvar or vaginal disease in the pooled FUTURE I and II studies after being randomised to and receiving at least one dose of HPV vaccine or placebo. The incidence rates for subsequent disease were calculated with case counting starting 60 days after cervical surgery or diagnosis of vulvar or vaginal disease.

Protocol specified guidelines were used to triage women with smear test abnormalities to colposcopy. 1 2 Colposcopists were trained to locate and biopsy all discrete abnormal areas on the cervix and lower genital tract. Loop electrosurgical excision was the preferred method for definitive therapy for cervical intraepithelial neoplasia grade II-III, adenocarcinoma in situ, and persistent cervical intraepithelial neoplasia grade I. However, other methods included cervix conisation, cryotherapy, and electrocauterisation. The frequencies of each technique recorded were loop electrosurgical excision (84.7%), cervical conisation (12.5%), cryotherapy (0.7%), and other (2.1%). As loop electrosurgical excision and cervix conisation accounted for 97.2% of the procedures performed, with no difference between the treatment groups (97.1% with placebo, 97.4% with vaccine) we use the term “cervical surgery” to describe any method used to treat cervical disease. Management of vulvar or vaginal disease followed the investigational site’s standards and practice and included surgical or topical therapy.

In both trials, participants were randomly assigned (1:1) to receive intramuscular injections of quadrivalent HPV vaccine (Gardasil or Silgard, Merck, Whitehouse Station, NJ) or visually indistinguishable placebo at day 1, month 2, and month 6. Comprehensive anogenital examinations and ThinPrep cervical cytology (Cytyc, Boxborough, MA, USA) were performed during scheduled visits, which occurred every 6–12 months after the vaccination phase. 1 2 Cytology specimens were classified according to the Bethesda System 2001. 12 Women reported any abnormality or suspected disease in between the scheduled study visits. All participants were required to use birth control during the vaccination phase (day 1 through to month 7), and all participants were evaluated for pregnancy before the administration of each dose of vaccine or placebo with a human chorionic gonadotropin assay sensitive to 25 international units. 13 Counselling about safe sex and contraceptive use was part of the study protocol.

The study designs and the results of the primary hypotheses have been described elsewhere. 1 2 Women were eligible to participate if they were not pregnant, did not report previous abnormal results on a cervical smear test, and had had a lifetime number of no more than four sex partners. Neither study included HPV testing (DNA or serology testing) or clinical examination before randomisation; thus, the trials allowed the enrolment of women with ongoing HPV infection or disease.

Between December 2001 and May 2003, 17 622 women aged 15–26 years were enrolled in one of two randomised, double blind, placebo controlled trials (FUTURE I and FUTURE II). 1 2 The studies were conducted in accordance with principles of Good Clinical Practice and were approved by the appropriate institutional review boards and regulatory agencies.

The primary objective of this retrospective analysis was to determine whether administration of quadrivalent HPV vaccine, compared with placebo, reduced the incidence of subsequent HPV related disease among women who had undergone surgery for cervical disease or who were diagnosed with vulvar or vaginal disease (genital warts, vulvar intraepithelial neoplasia, or vaginal intraepithelial neoplasia) after enrolment into the FUTURE I or FUTURE II clinical trials (fig 1 ⇓ ). Subsequent HPV related disease is defined as any disease that was detected at least 60 days after treatment or diagnosis. We measured the vaccine’s impact on end points that were associated with HPV types 6, 11, 16, and 18 specifically and on end points irrespective of causal HPV type.

In the previous analyses, we considered the impact of vaccination collectively among women who were diagnosed with genital warts, vulvar intraepithelial neoplasia grade I or worse, or vaginal intraepithelial neoplasia grade I or worse. If we consider only those women who were diagnosed with genital warts, there were 134 vaccine and 351 placebo recipients, respectively. The average time to the first detection of genital warts in these women was 1.4 years in the vaccine group and 2.1 years in the placebo group. Of these women, 43.3% (58/134) vaccine recipients and 14.8% (52/351) placebo recipients were infected with HPV types 6 or 11 at study entry. When we followed these women for recurrent genital warts, vaccination was associated with less recurrence of genital warts related to vaccine HPV types by 46.8% (10 cases in vaccine group v 33 cases in placebo group), but the reduction was not statistically significant.

Vaccination was associated with a reduced risk of any subsequent HPV related disease, irrespective of causal HPV type, by 35.2% (13.8% to 51.8%) (table 4 ⇓ ). For end points related to vaccine HPV types, there was a 64.4% reduction (41.6% to 79.3%) in any disease observed. Fig 4 ⇑ shows the divergence of incidence rates over time for any subsequent disease, and any subsequent cervical disease, irrespective of causal HPV type. Among both vaccine and placebo groups, the incidence of any subsequent disease (fig 4C) and any subsequent cervical disease (fig 4D) had not reached a plateau by end of study. Vaccine efficacy estimates were similar in the sensitivity analysis which used a 90 day interval between initial surgery or disease diagnosis and subsequent disease diagnoses (supplementary table 2 on bmj.com).

Fig 4 ⇓ shows the divergence of incidence rates over time for any subsequent disease and any subsequent vulvar or vaginal disease, irrespective of causal HPV type. Among both vaccine and placebo recipients, the incidence of any subsequent HPV related disease (fig 4A) had not reached a plateau by the end of study, and the divergence of incidence between the vaccine group and the placebo group increased at each six month interval. In contrast, the incidence of subsequent vulvar or vaginal disease after cervical surgery seemed to have reached a plateau for both vaccine and placebo recipients by end of study (fig 4B).

Table 3 ⇓ shows the vaccine efficacy for cervical intraepithelial neoplasia grade I or worse due to 10 high risk, non-vaccine HPV types after surgery for cervical disease. Vaccination was associated with reduction of cervical intraepithelial neoplasia grade I or worse associated with HPV31, 33, 45, 52, 58 (the five most common HPV types found in cervical cancer after HPV16 and HPV18) by 56.6% (3.4% to 82.3%). For individual HPV types, a significant reduction was observed for HPV31 (88.4%, [18.6% to 99.7%]). A positive percent reduction was observed for seven of the other types analysed, though the data did not reach statistical significance.

As shown in table 2 ⇑ , two vaccine and nine placebo recipients developed cervical disease related to vaccine HPV types after their first cervical surgery. Of the two women in the vaccine arm, one developed cervical intraepithelial neoplasia grade I and II lesions related to HPV type 16 after surgery. This woman was DNA positive to HPV types 16, 31, 56, and 58 at study entry and had HPV types 16 and 58 detected in her original surgical specimen. The second woman developed a cervical intraepithelial neoplasia grade I lesion related to HPV 18. She was DNA positive to HPV 18 at day 1 (and was negative to the other 11 tested high risk HPV types) but underwent electrocauterisation, so no surgical specimen existed. Of the nine women in the placebo arm who developed cervical disease related to vaccine HPV types, six (67%) did not have the same vaccine HPV type detected in their original surgical specimen.

Vaccination was associated with a significantly reduced risk of any subsequent HPV related disease after cervical surgery, irrespective of causal HPV type, by 46.2% (95% confidence interval 22.5% to 63.2%) (table 2 ⇓ ). Vaccination was associated with a significantly reduced risk of any subsequent cervical disease (by 48.3% (95% confidence interval 19.1% to 67.6%) for cervical intraepithelial neoplasia grade I or worse) and any subsequent high grade cervical disease (64.9% (20.1% to 86.3%) for cervical intraepithelial neoplasia grade II or worse). A significant reduction in the incidence of genital warts was observed (63.0% (10.3% to 86.6%)). Vaccination was also associated with significantly reduced risk of any subsequent disease related to vaccine HPV types (79.1% (49.4% to 92.8%)). The impact on vulvar or vaginal disease was primarily driven by a reduction in the incidence of genital warts. Vaccine recipients saw an 89.0% reduction (54.9% to 98.7%) in genital warts related to vaccine HPV types after cervical surgery. Vaccine efficacy estimates were similar in the sensitivity analysis which used a 90 day interval between the first and subsequent disease diagnoses (see supplementary table 1 on bmj.com).

Placebo recipients who were treated for HPV related disease in these clinical trials were at increased risk for developing subsequent HPV related disease (fig 3 ⇓ ). Within an average of only 1.3 years after cervical surgery (maximum follow-up of 3.7 years), the incidence of any subsequent disease among placebo recipients was 12.2 per 100 person years at risk, and 5.2% (31/593) developed subsequent high grade cervical, vulvar, or vaginal disease (incidence of 3.7). Compared with those who underwent cervical surgery, those who were diagnosed with genital warts, vulvar intraepithelial neoplasia, or vaginal intraepithelial neoplasia had nearly three times the risk for developing any subsequent HPV related disease (incidence of 31.0). Within an average of only 1.2 years after the initial diagnosis of vulvar or vaginal disease (maximum follow-up of 4.0 years), 13.0% (55/422) developed high grade cervical, vulvar, or vaginal disease (incidence of 8.4). For both analysis populations, the incidence of subsequent low grade disease (genital warts; vulvar or vaginal or intraepithelial neoplasia grade I; or cervical intraepithelial neoplasia grade I) was also high (10.1 to 26.1).

As expected, more placebo recipients underwent cervical surgery for disease due to any HPV type (n=763) than vaccine recipients (n=587). Among women who underwent cervical surgery, vaccine recipients had numerically higher baseline (at day 1 of the study) prevalence of squamous intraepithelial lesions (36.5%) compared with placebo recipients (30.0%) and higher prevalence of HPV DNA (70.1%) than placebo (62.0%) (table 1 ⇓ ). Among women who had a diagnosis of genital warts, vulvar intraepithelial neoplasia, or vaginal intraepithelial neoplasia, vaccine recipients also had a higher baseline prevalence than placebo recipients of squamous intraepithelial lesions (27.0% v 14.2%) and HPV DNA (65.9% v 44.0%) and a lower proportion of current or former smokers (36.7% v 43.4%).

The FUTURE I and II trials collectively enrolled and randomised 17 622 women. The populations analysed to determine the impact of quadrivalent HPV vaccine on subsequent disease is shown in fig 2 ⇓ . Of the 1350 women included in the analysis, only five (three placebo recipients, two vaccine recipients) received fewer than all three doses, with one woman in the placebo group having only one dose, and the others receiving two doses each. These women stopped the vaccination phase because pregnancy (two women), lactation (one), medical history (one), and unknown reason (one), but continued the follow-up phase of the study.

Discussion

Our results of the subgroup analysis demonstrate that vaccination with the quadrivalent HPV vaccine was associated with reduced incidence of subsequent cervical, vulvar, and vaginal intraepithelial neoplasia and genital warts in women who had been diagnosed and treated for cervical and vulvar or vaginal disease. After treatment for high grade cervical intraepithelial neoplasia, women are at increased risk for cervical cancer in the long term,19 and our data show they are at risk for subsequent diagnoses of premalignant disease in the short term. Within two years after treatment, those with prior vaccination had significantly reduced risk of subsequent high grade cervical disease. Our analysis also shows that for women who were diagnosed with HPV related vulvar or vaginal disease, those with prior vaccination had significantly reduced risk of developing any subsequent HPV related disease as well as disease related to the vaccine HPV types (types 6, 11, 16, and 18). These are, to our knowledge, the first results in vaccinated women who have undergone treatment for HPV related diseases.

Strengths of study We took several steps to enhance the accuracy, reproducibility, and generalisability of our findings. We considered all disease end points, irrespective of causal HPV type. Histological diagnoses were determined by a panel of expert gynaecologic pathologists who were unaware of women’s treatment or history of cervical or vulvar disease. Generalisability was enhanced by enrolling women from both developed and developing nations and by using standard management algorithms for treatment of cervical disease and local standards of care for treatment of vulvar or vaginal disease.20 21 In addition to measuring the impact on precancerous lesions, we also measured the impact on low grade disease. Although low grade cervical and vulvar or vaginal lesions are considered to be a morphological manifestation of an HPV infection, these lesions contribute to the clinical and economic burden of HPV disease. For example, the psychosocial22 23 24 and economic25 26 implications of genital warts are substantial and reflect, in part, the high transmission and recurrence rates.27 28 29 Lesion and infection are a local phenomenon of the transformation zone at the cervix, which is completely removed by treatment, whereas vulvovaginal disease is a field infection (infectious areas are not completely removed by the treatment and, therefore, the recurrence rate is high). In contrast to breast and cervical cancers, there are no screening programmes for vaginal and vulvar malignancies, and precursors of vulvar and vaginal cancers are often not recognised.7 Cervical, vulvar, and vaginal intraepithelial neoplasia grade I and genital warts have also been shown to contain a variety of high risk HPV types.6 30 31 32 33 In this study, the highest rates of subsequent disease observed was low grade, and vaccination rapidly and significantly reduced the onset of subsequent low grade lesions, particularly among women who underwent cervical surgery.

Comparison with other studies These data are in line with previous studies that suggest the benefits of vaccination are not limited to the primary target group of young, sexually naïve girls. In an intention to treat analysis that measured the impact of the quadrivalent HPV vaccine in a mixed population of HPV naïve and HPV infected women, vaccination significantly reduced the number of abnormal smear test results and procedures such as colposcopy, biopsy examination, and definitive therapy irrespective of the causal HPV type.34 Another study has shown the quadrivalent HPV vaccine generates an anamnestic response (renewed rapid production of an antibody on a subsequent encounter with the same antigen) in women aged 15–26 who are seropositive before vaccination,35 36 and that the quadrivalent HPV vaccine prevents reinfection or reactivation of disease that is related to vaccine HPV types.37 For example, women who had cleared an HPV16 infection in the past were protected from developing subsequent HPV16 related disease.37 A previous study has shown that women who are infected with one or more vaccine HPV types derive residual benefit by the quadrivalent HPV vaccine’s prevention of infection and disease from HPV type(s) to which the woman has not yet been exposed.38 The vaccine is also effective in women up to the age of 45, whereby prophylactic vaccine efficacy against disease related to vaccine HPV types was 92.4% (49.6% to 99.8).39 40

Limitations of study This retrospective analysis is accompanied by some limitations. The trials were not designed or powered to evaluate the effects of vaccination after cervical surgery or diagnosis. Women with a prior history of HPV related disease were excluded from enrolment, so we cannot directly measure the vaccine’s impact in women who have undergone treatment before vaccination, since all women in this study were vaccinated before treatment. As a surrogate, we identified women who were treated for HPV related disease after randomisation and followed them for additional disease outcomes. In this relatively low risk population (healthy women with a mean of two lifetime sexual partners and no prior history of HPV related disease) the risk of subsequent disease after a diagnosis of HPV related vulvar or vaginal disease was about three times higher than after cervical surgery. Although the reasons for this are unclear, our data indicate that women who were diagnosed with HPV related disease were a high risk group at study entry, as nearly half of these women (44–70%) were infected with at least one of the 14 tested HPV types, and 14–37% had abnormal cytology at day 1. There was an imbalance in some baseline characteristics (that is, at day 1) as a result of our selection of patients who had treatment for cervical or vulvovaginal disease after vaccination that may have potentially biased these results. Among women who had a pathology panel diagnosis of vulvar or vaginal disease, placebo recipients had a higher proportion of current or former smokers (43.4%) compared with vaccine recipients (36.7%), and women who smoke are about twice as likely as non-smokers to get cervical cancer.41 However, the most important risk factor for cervical cancer is infection by HPV,41 and, because of our subsetting to those who later received treatment for cervical or vulvovaginal disease, vaccine recipients had numerically higher day 1 prevalence of HPV DNA and day 1 prevalence of squamous intraepithelial lesions compared with placebo recipients. In spite of these potential biases, there were still significant reductions in subsequent disease for vaccine recipients compared with placebo recipients. It is important to note that most of the “first” disease detected in both vaccine and placebo recipients was a result of these infections that were present at day 1, and not due to vaccine failure. It is also important to note that the impact of the vaccine in preventing subsequent disease does not indicate that vaccination provides a therapeutic effect. Though it is possible that some of the cervical disease detected after cervical definitive therapy is residual, these data suggest that most of the subsequent disease is from an infection with an HPV type that was not the causal type of the first lesion. For example, we found that six of the nine women in the placebo arm who developed cervical disease related to vaccine HPV types after surgery had a vaccine HPV type that was not detected in the original surgical specimen, suggesting that the lesion diagnosed after surgery was from a new infection and not from residual disease. Four of the six women in the placebo group who developed cervical intraepithelial neoplasia related to a vaccine HPV type after surgery that was not in the original surgical specimen also reported at least one new sexual partner in the interval between the first cervical surgery and the subsequent disease diagnosis. We have also previously shown that vaccination of women who were negative to 14 HPV types at enrolment reduced the incidence of cervical intraepithelial neoplasia grade III or worse associated with 10 non-vaccine HPV types by 32.5% (6.0% to 51.9%), and the present analyses suggest some of the subsequent disease that is prevented is due to protection against HPV types that are phylogenetically related to vaccine HPV types (6, 11, 16, and 18).42 Although the exact underlying mechanism is not fully known, these observed reductions are felt to be clinically significant.