Efficacy of a bivalent HPV 16/18 vaccine against anal HPV16/18 infection among young women: a nested analysis within the Costa Rica Vaccine Trial

Introduction

Anal cancer remains rare, with annual age-standardized incidence rates in the general population of ∼1.5/100,000 for women (1); but, rates have been approximately doubled over the past several decades in many countries, including the US and several European nations (2-5). The absolute burden of anal cancer is higher for women than men (3-5), yet, anal cancer disproportionately affects HIV-positive individuals and men who have sex with men (MSM), even if they are HIV-negative (6, 7).

Human papillomavirus (HPV) causes the majority of anal cancers, with an estimated 75 to 80% of HPV-associated anal cancers caused by HPV type 16 or 18 (8; 9). For other HPV-associated extra-cervical cancers, including cancers of the oropharynx, vagina, vulva, and penis (9), variable proportions are caused by HPV infection but, where HPV is involved, HPV16 is the vastly predominant type implicated in the cancer (range 75% to 95% of the HPV-associated cancers) (9).

Two vaccines prevent HPV types 16 and 18 infections: the bivalent HPV16/18 vaccine (Cervarix®*, GlaxoSmithKline Biologicals)(10) and the quadrivalent HPV 6/11/16/18 vaccine (Gardasil™, Merck and Co, Inc,) (11). Among women, vaccine efficacy (VE) against cervical (both vaccines) (10, 11) and vaginal and vulvar (quadrivalent only) (12) HPV infections and related diseases has been demonstrated. For men, VE against penile, perianal, perineal HPV infections as a combined endpoint has been demonstrated (quadrivalent only) (13). Direct evidence for efficacy against anal HPV infection and disease is lacking with the exception of one unpublished trial of the quadrivalent vaccine among ∼600 MSM (14). The bivalent vaccine has not been evaluated at extra-cervical sites.

We conducted a study to evaluate efficacy of the bivalent HPV vaccine to reduce anal HPV infection, nested in a community-based randomized cervical vaccine efficacy trial of adult young women.

Methods

Women included in the present evaluation are participants in a randomized clinical trial initially designed to evaluate the efficacy of a bivalent HPV vaccine against persistent type-specific HPV16/18 infection and associated precancerous lesions at the cervix (15, 16). The study enrolled women residing in Guanacaste and selected areas of Puntarenas, Costa Rica, identified via a census, between 2004-2005. Main eligibility requirements were: age 18-25 years, planned residence in the area for the 6 months following enrollment, in good general health, neither pregnant nor breastfeeding, and willing to provide written informed consent. Women were excluded if they had preexisting medical conditions that required chronic treatment or caused immunosuppression, had a history of hepatitis A or previous vaccination against it, or were unwilling to use contraception during the vaccination period. 7,466 women were enrolled; they represented 59.1% of eligible and 30.5% of all (eligible plus ineligible) women from the census (15). The trial was approved by the human subjects review committees of the United States (US) National Cancer Institute and INCIENSA (Instituto Costarricense de Investigación y Enseñanza en Nutrición y Salud) in Costa Rica.

At the enrollment visit (following informed consent and risk-factor interview), a pelvic examination was performed on sexually experienced women, exfoliated cervical cells were collected in PreservCyt medium (Cytyc Corp, now Hologic, Marlborough, Massachusetts) for Thinprep (Cytyc Corp) cytologic evaluation and HPV DNA testing, and blood was collected for HPV16/18 serology. Next, women were randomized in a double-blinded fashion to receive the HPV vaccine (Cervarix, GlaxoSmithKline Biologicals) or a “Control” hepatitis A vaccine (a modified preparation of Havrix, GSK Biologicals). Both vaccines were formulated in three 0.5 ml doses with identical packaging. HPV and control vaccines were assigned random vaccine identification numbers at the time of labeling by the manufacturer. Study personnel at the Costa Rican study site randomized participants by assigning each eligible participant to the next available sequential vaccine identification number. The protocol called for a dose of vaccine at each of 3 study visits: at enrollment, 1 month following the initial dose (allowable range, 21-120 days), and 6 months following the initial dose (allowable range, 121-300 days). Women not attending their visits in the allowable range for the 2^nd dose (21-120 days after enrollment) remained eligible for the final dose; women who missed the window for the final dose (121-300 days after enrollment) did not receive that dose (15).

At annual follow-up visits, clinicians collected from sexually active women exfoliated cervical cells (same method as above) for cytologic evaluation and HPV DNA testing Women with low-grade cytologic abnormalities were evaluated every six-months until 3 consecutive normal cytologic results returned them to yearly follow-up. Women with cervical high-grade disease or persistent low-grade abnormalities were referred to cervical colposcopy for evaluation and treatment if needed.

Sampling of the anus was introduced at the four-year study visit, the final blinded study visit of the trial. At this study visit, a questionnaire was administered that included questions on anal sexual behaviors; while some under-reporting of stigmatized behaviors such as anal sex is expected, we don't anticipate differential reporting by vaccination status that would bias our efficacy estimates.

The anal specimen was collected prior to the pelvic exam among sexually active women (defined by vaginal intercourse) by inserting a dry swab 3-4 cm into the anal canal, rotating one time, and then removing the swab while rotation continued using gentle pressure against the wall of the anal canal. The swab was placed in 1ml of PreservCyt and frozen immediately in liquid nitrogen. While the predictive value of one-time detection of anal HPV16 or 18 likely for anal precancer or cancer is likely very low, and there are not standard clinical recommendations for need of follow-up in such cases, a subset of women with anal HPV16/18 infection will be monitored during the long-term follow-up phase of the trial. Most women will be followed up to 10 years since initial vaccination; as part of this effort, long-term type-specific anal HPV persistence and related disease will be monitored, and clinical management will be provided if necessary.

Anal and cervical samples were tested for HPV DNA using the SPF₁₀ PCR primer system and a DNA enzyme immunoassay detection of amplimers (DEIA; DDL Diagnostic Laboratory [Voorburg, the Netherlands]); if positive, genotyping was conducted using the line probe assay (LiPA₂₅) (SPF₁₀PCR/LiPA₂₅ HPV genotyping assay system, version 1, Labo Bio-medical Products, Rijswijk, the Netherlands) (17). LiPA₂₅ detects 25 HPV genotypes, including carcinogenic (HPV16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68/73) and non-carcinogenic (6, 11, 34, 40, 42, 43, 44, 53, 54, 66, 70, and 74) types. To ensure that HPV16 and HPV18 infections were not missed, all positive specimens on SPF₁₀ PCR/DEIA that were negative for HPV16 or HPV18 by LiPA₂₅ were also tested using HPV16 or 18 type-specific primers (18, 19).

Serum collected at enrollment was used to determine HPV16 and HPV18 serological status using a VLP-based direct ELISA, a standard measure that measures polyclonal antibodies (GlaxoSmithKline Biologicals, Rixensart, Belgium), as described previously (20, 21). Antibody results were dichotomized using standard cutoff points calculated from antibody titer values 3 SDs above the geometric mean titers taken from a group of HPV-negative individuals (20). Cut points were optical density of at least 8 EU/mL for anti-HPV16 and at least 7 EU/mL for anti-HPV18 (20, 21).

Results

Of the 7466 women randomized (CONSORT), 6352 attended the four-year study visit (3181 HPV; 3171 Control). 1744 (873 HPV; 871 Control) women refused anal specimen collection and virgins (n=384) were not eligible; the acceptance rate of anal specimen collection among eligible women was 70.8% (4224 out of 5968). After excluding 14 inadequate anal specimens, the full analytic cohort comprised 4210 women (2103 HPV; 2107 Control). Median follow-up time was 48.8 months (4.1 years) and was similar between arms (HPV 48.9 months [range 39.5–74.5 months]; Control 48.8 months [range 38.7-74.5 months]; p=0.9). The restricted cohort of women who were negative for cervical HPV16/18 DNA and serology at enrollment included 1989 women (1003 HPV; 986 Control).

Percentages of women who accepted specimen collection were the same in both arms (70.8% for both HPV [2112 out of 2985] and Control [2112 out of 2983]). Women who provided an anal specimen were older (46.6% [1970/4224] vs. 39.9% [695/1744] in the older age category at enrollment [22 to 25 yrs], p<0.001), more likely to have cervical HPV16 positivity at enrollment (7.2% [305/4224] vs. 5.2% [90/1744], p<0.001), more likely to have 4+ lifetime sexual partners (38.0% [1604/4224] vs. 31.3% [546/1744], p<0.001), and more likely to report anal sex (22.3% [944/4224] vs. 8.2% [143/1744], p<0.001) than women who declined.

Distributions of pre- and post-randomization characteristics were similar in the HPV and control arms, including age at entry, enrollment cervical cytology, cervical HPV DNA positivity, and HPV seropositivity and self-reported anal sex (Table 1).

In the restricted cohort, VE against HPV16/18 infection detected at the anus four-years following administration of the first vaccine dose was 83.6% (95%CI 66.7% to 92.8%; 8 and 48 events in the HPV and control arms, respectively), which was comparable to the cervical HPV16/18 VE among the same women from specimens collected at the same time point (87.9%, 95%CI 77.4% to 94.0%; 10 and 81 events in the HPV and control arms, respectively).

In the full analytic cohort, VE against HPV16/18 infection detected at the anus four-years post-vaccination was 62.0% (95%CI 47.1% to 73.1%; 47 and 124 events in the HPV and control arms, respectively); the corresponding cervical VE was 76.4% (95%CI 67.0% to 83.5%; 40 and 170 events in the HPV and control arms, respectively) (p for anatomic-site interaction =0.031) (Table 2). VE against anal HPV16 was 68.2% (95%CI 51.4% to 79.7%; 27 and 85 events in the HPV and control arms, respectively) and against anal HPV18 was 55.5% (95%CI 25.2% to 74.2%; 20 and 45 events in the HPV and control arms, respectively); VE against cervical HPV16 was 75.8% (95%CI 63.8% to 84.2%; 28 and 116 events in the HPV and control arms, respectively) and cervical HPV18 was 78.6% (95%CI 62.0% to 88.7%; 13 and 61 events in the HPV and control arms, respectively) (p for anatomic-site interaction: HPV16=0.3; HPV18=0.05).

In the full cohort, significant and comparable cross-protection against a composite endpoint of infection with HPV31/33/45 was observed at the anus (VE 49.4%, 95%CI 30.3% to 63.6%; 55 and 109 anal events in the HPV and control arms, respectively) and the cervix (VE 45.2%, 95%CI 27.7% to 58.7%; 76 and 139 cervical events in the HPV and control arms, respectively) (p for interaction = 0.65) (Table 3). Individually, significant VE was present against HPV 31 and 45 but not 33 (Supplementary table 1). The results for VE against heterologous HPV types observed in the full cohort were similar to the results in the restricted cohort (Table 2 and Supplementary table 1). No VE was observed for all other carcinogenic HPV types (after excluding 16/18/31/33/45) (full cohort: anal VE -3.2% [95%CI -18.6% to 10.1%], 405 and 393 anal events in the HPV and control arms, respectively; cervical VE 3.9% [95%CI -9.4% to 15.6%], 445 and 464 cervical events in the HPV and control arms, respectively).

At the final study visit when anal sex was queried, women in the full cohort who reported anal sex had an anal VE of 73.9% (95%CI 52.7% to 86.4%) while the anal VE among women who did not was 55.3% (95%CI 33.5% to 70.4%; p for interaction by anal sex status =0.13); cervical VE was comparable by reported anal sex status (77.5% [57.4% to 88.8%] and 76.2% [64.7% to 84.3%], respectively, p for interaction by anal sex status =0.89) (Table 4). In the restricted cohort, anal and cervical VE estimates were comparably high, regardless of anal sex (Supplementary table 2).

Discussion

A randomized analysis of data from our community-based HPV vaccine trial in Costa Rica demonstrates that the bivalent HPV vaccine is efficacious against prevalent anal HPV 16 and 18 infections in young women measured four years following vaccination, and that the protection is likely comparable to that observed for cervical HPV infections. We also show the first evidence of cross-protection against a composite endpoint of HPV types 31, 33 and 45 at an extra-genital site; providing confirmation that the protection afforded by the bivalent HPV vaccine goes beyond the HPV types included in the vaccine formulation.

Our estimate of anal HPV16/18 VE for the bivalent vaccine among our full analytic cohort (62%) is similar to the only other study of the efficacy HPV vaccine against anal infection/related disease. There, the VE of the quadrivalent vaccine against anal HPV 16/18 infection and related lesions was 50% among ∼600 (115 total events) HIV-negative MSM in their full analytic cohort (age 16-26 years, median follow- up time: 32 months) (14).

Anal HPV VE among women who reported anal sex, and are therefore at significantly increased risk for anal cancer (26), appeared higher than efficacy among women who reported no anal sex in the full cohort. One possibility may be that a greater proportion of the anal “infections” detected among women who did not have anal sex may be superficial virions shed from genital sites for which the vaccine would not be expected to protect. As estimates of anal VE were similar by report of anal sex in the restricted cohort, vaccination before exposure appears to be highly efficacious in all subgroups of women.

The main limitation of this analysis is that only one anal specimen was collected, 4 years post vaccination. As such, we were unable to assess anal HPV before vaccination, or use HPV incidence or persistence instead of prevalence as our endpoint. Estimated VE is reduced by inclusion of women with prevalent infection at the time of vaccination, not protected by this prophylactic vaccine (16). Persistent infection is a preferred trial endpoint over one-time detection because it reduces measurement error, is associated with higher absolute risk of anal cancer, and is therefore a better proxy for cancer prevention. Thus, determining VE against persistent anal HPV infection and associated lesions is necessary among women. Further, demonstrating this protection lasts beyond the four years demonstrated in this study is critical.

Our study is likely to have internal validity because: it is randomized, the majority of women agreed to specimen collection, and refusal to have specimens collected was not differential by arm. Less certain is the external validity of findings, but this should be couched in the context that phase III trials are typically not externally valid and that our trial is, if anything, more likely to be generalizable since enrolment was based on a true census of a defined geographical area (i.e., a true community-based trial).

The HPV vaccines have great potential for preventing a large proportion of HPV-associated cancers at the anus and other anatomic sites, assuming adequate duration of protection. Among women, there is published evidence for VE against HPV16/18 infections at the cervix, vagina, vulva (10-12), and now the anus, while for men, protection has been demonstrated at extra-genital and anal anatomic sites (13). While VE against oral HPV has not been demonstrated, vaccination might also prevent most HPV-associated oropharyngeal cancers. The implications that 1) HPV causes extra-cervical cancers, and 2) the vaccine protects against the infections that cause these cancers, differs between countries with and without effective cervical cancer screening programs. Countries without cervical cancer screening have high rates of cervical cancer and the absolute burden of HPV-associated cancers will remain many times higher for cervix than the combined non-cervical sites, implying that limited resources for vaccination should be focused on women and not men. However, high-resource countries with cervical cancer screening typically have dramatically diminished rates of cervical cancer. The use of HPV vaccines among males might be affordable and useful because the number of HPV-associated cancers might end up being greater for men, since rates of anal and oropharyngeal cancers are increasing and the latter is predominantly diagnosed among men (27).

This first evaluation of the vaccine efficacy against anal HPV in women suggests that the bivalent HPV vaccine protects against the majority of anal HPV16/18 infections and that the overall anal VE was comparable to that of cervical VE. The cumulative findings to date suggest that the rate HPV-associated cancers at many anatomic sites will be greatly reduced among women who receive the prophylactic HPV vaccines prior to exposure.

Supplementary Material