https://orcid.org/0000-0003-2913-1736
ABSTRACT
The main aim of our study was to investigate the specific contribution of a 9-valent human papillomavirus vaccine (9vHPV) to the recurrence risk of cervical intraepithelial neoplasia grade 2 or worse (CIN2+) in women vaccinated post-excision. Therefore, we conducted a retrospective monocentric cohort study in women aged 22–49 years undergoing conization between 2014 and 2023. The 9vHPV-vaccinated women were matched to unvaccinated women for age and follow-up duration in a 1:2 ratio to eliminate allocation bias. The risk of CIN2+ recurrence was estimated by the incidence rate ratio using Poisson regression with adjustment for comorbidities, smoking status, nulliparity, CIN grade, positive cone margin, and HPV genotypes. The CIN2+ recurrence rates in 147 women enrolled in the analysis were 18 and 2 cases per 100,000 person-days for unvaccinated and vaccinated women, respectively, during a mean follow-up period of 30 months (±22 months). A reduction in CIN2+ recurrences by 90% (95% confidence interval: 12–99%) was documented in 9vHPV-vaccinated participants compared to women undergoing only surgical excision. Moreover, vaccinated women with a positive cone margin showed a 42% (though non-significant) reduction in relapse (p = .661). Full post-conization vaccination with the 9vHPV contributed to an additional reduction in the risk of CIN2+ recurrence. This finding is consistent with current knowledge and suggests a high adjuvant effect of the 9vHPV vaccine.
Introduction
The benefit of post-excisional immunization with a human papillomavirus (HPV) vaccine against recurrences of cervical intraepithelial neoplasia grade 2 or worse (CIN2+), irrespective of a specific high-risk HPV genotype, was demonstrated in all 11 studies published between 2011 and 2023, as reviewed in the current meta-analysis.Citation1 HPV vaccination initiated on the day of conization or within one year of surgical excision could reduce the risk of CIN2+ recurrence by 67–85% over the course of up to 6 years of follow-up.
This conclusion was primarily based on estimated effects of bi- or quadrivalent HPV vaccines, which have been available since 2006. However, a 9-valent HPV vaccine (9vHPV) under the brand name Gardasil 9 has been in worldwide use since 2014. This vaccine targets not only the most prevalent high-risk HPV genotypes (16 and 18) but also five other high-risk genotypes: 31, 33, 45, 52, and 58. Importantly, this multi-valent, 9vHPV vaccine has shown promise for greater beneficial impact in both men and women, as suggested by recent studies.Citation2,Citation3
While the 9vHPV vaccine offers the promise of additional impact on the reduction in CIN2+ recurrences, its adjuvant effect has thus far only been investigated alongside lower-valent vaccines in two studies.Citation4,Citation5 Only one current study assessed the exclusive effectiveness of the 9vHPV vaccine in women with mixed-start vaccination (initiated both prior to and after conization).Citation6 The outcomes of these three studies suggest a favorable adjuvant effect of the 9vHPV vaccine, which aligns with the findings of studies involving only lower-valent HPV vaccines. However, additional contributions of the other five high-risk genotypes contained in this vaccine have not yet been investigated.
There is limited understanding of why HPV vaccination performed after excision could enhance the adjuvant effect. Conization could regulate pro-inflammatory cytokines induced by local HPV infection, as well as trigger a low degree of local inflammation, thus supporting a stronger immune response to vaccination.Citation7–9 These mechanisms could possibly explain the favorable effects of post-conization vaccination.
To date, no study designed to assess the effect of post-excision vaccination with only the 9vHPV vaccine on the reduction of CIN2+ recurrence in women after surgical excision has been conducted. We therefore performed this cohort study with eligible women – both those never vaccinated or receiving the 9vHPV vaccine after conization.
Material and methods
Study population
This retrospective cohort study was conducted at the Center of Ambulatory Gynecology and Primary Care (Brno, Czech Republic), where a total of 649 women underwent at least one conization between January 2014 and August 2023. Only women who met the following inclusion criteria were enrolled: a) age 18 years and older; b) primary conization for histologically confirmed CIN2+; and c) conization performed using the loop electrosurgical excision procedure (LEEP). Women with CIN2+ recurrence confirmed within ≤6 months of primary conization, those who received a non-9vHPV vaccine, were previously vaccinated, or started vaccination before conization, were excluded from the study. Women with a short-term follow-up of less than 6 months were also ineligible for this study. Anonymized clinical and demographic data were obtained from the patient registry of the Center of Ambulatory Gynecology and Primary Care and the central laboratory of Unilabs Pathology (Prague, Czech Republic).
Study design
The main objective of this study was to assess the effectiveness of post-excision vaccination with a 9vHPV against CIN2+ recurrence, regardless of the HPV genotype, for more than 6 months after conization. The study endpoint was the histologically proven presence of CIN2+, followed by re-conization. The first follow-up visit, scheduled for 6 months after excision, involved a Pap smear, HPV testing, and a colposcopy, with a colposcopy-directed biopsy if indicated. Regular Pap smears and colposcopies were conducted every year. If indicated, the examinations were extended by additional HPV testing including biopsy.
The need for excision was documented not only by cytological but, also by histological diagnosis. The CIN finding was stratified into grade 2 or grade 3 or worse, including carcinoma. The presence of high-risk HPV genotypes was determined using the Cobas HPV test (Cobas 4800 System; Roche Molecular Diagnostics, Switzerland). This method detected 14 high-risk HPV genotypes and – in addition to the other 12 genotypes (HPV 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68) – provided specific genotyping for HPV 16 and 18. The excisional biopsy specimens were thoroughly examined, and either positive or negative cone margin status was determined. Women with a positive finding underwent cytology at 4–6 months after treatment in accordance with international guidelines.Citation10
Study variables included age (relative to conization date), smoking status, comorbidities, nulliparity status, follow-up duration, and vaccination status. Female smokers were classified as either current or former, while nonsmokers had never smoked. If the record on smoking was missing, smoking status was classified as unknown. Follow-up duration was determined by the date of either repeat conization or the last visit within the study period.
The study was approved by the Ethics Committee of the Third Faculty of Medicine, Charles University in Prague. The final report of this cohort study followed the Strengthening the Reporting of Observational Studies in Epidemiology guidelines.Citation11
Statistical analysis
Categorical variables, presented as absolute numbers and percentages, were compared using the χ2 or Fisher exact test. Continuous variables were reported as mean and standard deviation (SD), median and interquartile range (IQR). To compare baseline characteristics between vaccinated and unvaccinated women after conization, standardized differences were used, with a meaningful difference considered to be > 10%.Citation12
Moreover, the vaccinated cohort was exactly matched to unvaccinated individuals using age and post-conization follow-up duration in a 1:2 ratio to minimize allocation bias. The Kaplan-Meier estimator was used to determine the cumulative incidence of CIN2+ recurrence.
The crude incidence rate ratio (IRR), including a 95% confidence interval (95% CI), was calculated from the incidence rates (IR) of CIN2+ recurrence in vaccinated versus unvaccinated matched women. Furthermore, Poisson regression was applied to adjust the IRR for covariates representing possible confounding variables. The log-rank test was used to assess the equality of failure functions. The level of post-vaccination protection or reduction in recurrences was estimated from crude and adjusted IRRs using the following equation: 100% × (1–IRR). The power of the test was established from the comparison of rates of both cohorts using a negative binomial regression two-sided test.
All tests were two-tailed, and the level of significance was set at 0.05. Statistical analyzes and regressions were performed using Prism 10 (GraphPad Software, Inc., San Diego, CA, USA) and STATA/SE version 18 software (StatCorp, Lakeway Drive, TX, USA), respectively.
Results
Between January 2014 and August 2023, CIN2+ recurrences were evaluated in 415 women meeting the inclusion criteria for this retrospective study (). A total of 98 unvaccinated women were age- and follow-up-matched in a 2:1 ratio with 49 women immunized with the 9vHPV vaccine. The mean age of the 147 enrolled women was 33 ± 6 years, with an average follow-up duration of 30 ± 22 months. The vaccination was started on the day of conization or later, i.e., at a median of 24 days (IQR: 6–35 days) after surgical excision. All vaccinated women received three vaccine doses, except for only six (12.2%) who were incompletely immunized with two doses. Thus, overall, the vaccinated cohort can be considered fully immunized.
Figure 1. Flowchart of cohort study.
Baseline characteristics included smoking status, comorbidities (hypertension, thyroid disease, Crohn’s disease, and others), nulliparity status, type of cervical dysplasia of grade 2 or worse, positive cone margins, and high-risk HPV genotypes in the specimen. These characteristics before and after matching of both cohorts, including standardized differences, are reported in .
Table 1. Baseline clinical a demographic characteristic for eligible and matched cohorts by 9vHPV vaccination status.
During follow-up, CIN2+ recurrence requiring repeat conization was documented in 17 out of the 147 women. Re-conization for recurrence of CIN2 (7 women) and CIN3+ (10 women) was required after a median of 61 months (IQR: 27–88 months) from prime excision. The incidence rate of repeat conization reached 18 per 100,000 person-days in the unvaccinated cohort, while only one excision in the vaccinated women represented a rate of 2 per 100,000 person-days. Post-conization HPV vaccination reduced the risk of recurrence of high-grade lesions by 87% (95% CI: 19–100%) as indicated by a crude IRR of 0.13 (95% CI: 0.00–0.81). This result is also documented by the Kaplan-Meier curve (). Moreover, the 85% power of the test demonstrated sufficient strength of this finding. When adjusting the IRR for variables exhibiting a standardized difference > 10%, 9vHPV vaccination decreased this risk by 90% (95% CI: 12–99%), . This finding was confirmed by an additional analysis conducted in women with a median follow-up of ≥720 days, as the IRR of 0 (95% CI: 0–0.68) was consistent with the overall outcome.
Figure 2. Probability of CIN2+ recurrence in 9vHPV-vaccinated and unvaccinated women relative to time since conization.
Table 2. Poisson regression model to assess the relevant risk factors for CIN2 recurrence.
The presence of comorbidities and nulliparity, as recorded during follow-up, had no effect on the risk of CIN2+ recurrence. Non-significantly increased incidence of CIN2+ recurrences was observed in smokers, in women with evidence of any of the 12 HR HPV genotypes other than HPV 16 and/or 18, and in those with the first excision for a CIN3+ finding. The relapse rate was significantly higher in women with a positive cone margin, as shown by 28 events per 100,000 person-days compared to that in women with a negative cone margin (9 events per 100,000 person-days) regardless of vaccination status. This finding was confirmed by an adjusted IRR of 3.31 (95% CI: 1.21–9.05). Moreover, a total of 8 CIN2+ recurrences were reported in women with a positive cone margin. While only one relapse was documented in vaccinated women (incidence rate of 18 per 100,000 person-days), 7 events were found in unvaccinated cohort (incidence rate of 31 per 100,000 person-days). An IRR of 0.58 (95% CI: 0.01–4.49) suggested a relapse reduction by 42% in the vaccinated compared to unvaccinated women. However, due to the small sample size of women with a positive cone margin, we were unable to determine the statistical significance of the reduced recurrence rate.
Discussion
To the best of our knowledge, this was the first cohort study to assess the effectiveness of post-conization vaccination exclusively with the 9vHPV vaccine. The level of protection against CIN2+ recurrence of any high-risk HPV genotypes, achieved by post-excision vaccination, increased by 90% compared to unvaccinated women. Moreover, this result was supported by the > 80% power of the test. A lower but consistent adjuvant effect of 58–80% has been observed in recent studies with women vaccinated not only with the 9vHPV vaccine, but also, in those exclusively receiving the 9vHPV vaccine, regardless of administration timing relative to excision.Citation4–6 Moreover, the average follow-up duration of these studies varied within a range of 20–33 months, consistent with the mean 30-month follow-up time in our study. Therefore, it is highly likely that 9vHPV vaccination after excision did contribute to the additional reduction in recurrence rates.
Furthermore, vaccination in the above studies was initiated within 0–12 months of conization. In our study, 75% of women received their first dose of the 9vHPV vaccine within 35 days of conization. Given that 88% of women were fully vaccinated with three doses and all of them had received at least two doses, the vaccination in our study could be considered complete. Hence, the effect of incomplete 9vHPV vaccination could not be further analyzed. The outcome of our study was consistent with the finding of the most recent meta-analysis, demonstrating a significant impact of post-conization vaccination with any HPV vaccine available, resulting in a 78% reduction in CIN2+ recurrence.Citation1
In terms of methodology and analyzes, our study outcomes align with the findings of a study conducted in women immunized with a quadrivalent HPV (4vHPV) vaccine after conization.Citation13 The adjuvant effect of this vaccine, calculated from the hazard ratio adjusted for covariates, demonstrated a 65% reduction in CIN2+ recurrences, regardless of specific high-risk HPV genotypes, within 6–48 months of follow-up. This was observed in women with a mean age of 37 years who had undergone conization for CIN2+.
However, it remains inconclusive whether the 9vHPV vaccine could increase effectiveness. Other observational studies have reported reduction rates achieved with the 4vHPV vaccine ranging between 78 and 92%, with one randomized clinical trial even demonstrating a 98% non-significant reduction in relapses.Citation14–18 The reported variability in results is likely attributable to differences in the methodologies and analyzes employed across studies.
Our study revealed a higher incidence of CIN2+ recurrence in women with a positive cone margin compared to those with a negative finding, as documented by an incidence rate ratio of 3.3. A similar outcome, supported by an adjusted hazard ratio of 4.9, was observed in another study.Citation13 Whether HPV vaccination could decrease CIN2+ relapse rates in these women was not conclusively demonstrated by our study, as only an non-significant 42% reduction was found due to the limited sample size of women with positive cone margins. Future studies should therefore aim to assess the impact of HPV vaccination under these specific conditions.
Even though the results of our study demonstrated a highly beneficial effect of 9vHPV vaccination with a statistical power exceeding 80%, the sample size was limited. The applied methods of matching and adjustment contributed to a robust result, minimizing the influence of confounding factors in the study.
Admittedly, a limitation of the study was the absence of data on smoking status in more than 62% of the women and no information at all on body mass index. To mitigate the risk of bias resulting from undetected HPV genotypes reported in 40 women (27%), an adjustment was applied for this covariate. Given that human papillomavirus is sexually transmitted, the risk of reinfection could be influenced by the HPV status of sexual partners. Unfortunately, as sexual partner history was missing in the patient registry, this parameter could not be included as a confounding factor in the analysis. Moreover, we were not able to assess the impact of 9vHPV vaccination on the other seven vaccine-related genotypes as the laboratory tests did not allow for their specification. As a result, only the common genotypes 16 and 18, contained in the bi- and quadrivalent vaccines, were considered vaccine-related ones. Whether vaccination can provide an adjuvant effect against CIN2+ relapse of any high-risk genotype or only vaccine-related genotypes is not yet clear. It may therefore be reasonable to expand the secondary prevention strategy to include use of CG-based gels or other appropriate antivirals.Citation19
Conclusion
The outcomes of our retrospective cohort study affirm the robust adjuvant effect of the 9vHPV vaccine in fully immunized women after conization, resulting in a 90% reduction in CIN2+ relapses compared to those undergoing excision only. However, the impact of HPV vaccination on the decrease in recurrence rates in women with a positive cone margin after excision remained inconclusive due to the limited sample size of this patient subgroup. Future research should focus on determining the adjuvant effect in women with a positive cone margin and exploring the potential influence of additional HPV genotypes contained in the multivalent vaccine.
Author contributions statement
The study was conceived by MP and designed by MP, IKL, and PD. The data were acquired and collated by VDjr, VD, and DL, and analyzed by MP. Funding was arranged by PD. Administrative and material support was negotiated by VDjr. and RP. The manuscript was drafted and revised critically for important intellectual content by all authors (VDjr, MP, VD, DL, PD, IKL, and RP). All authors gave final approval of the version to be published. MP, VDjr, and VD had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.
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Data availability statement
The data that support the findings of this study are available from the corresponding author upon reasonable request.
Supplementary material
Supplemental data for this article can be accessed on the publisher’s website at https://doi.org/10.1080/21645515.2024.2343552.
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References
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