Methods

Women included in the present evaluation were participants in a larger community-based randomized clinical trial of 7466 Costa Rican women 18−25 years of age at enrollment (June 2004 to December 2005) that evaluated the efficacy of the HPV-16/18 virus−like particle vaccine formulated with the AS04 adjuvant system (Cervarix; GSK Biologicals, Rixensart, Belgium) against persistent type-specific infection with HPV and HPV-associated precancerous lesions. The design and methods for this trial have been described in detail previously and involved randomization, vaccination (3 doses offered over 6 months), active annual (or more frequently if clinically indicated) follow-up, referral to colposcopy of high-grade precancers and/or persistent low-grade cervical lesions, and treatment by loop electrosurgical excisional procedure (LEEP) when clinically indicated.

For the present analyses, we included 2 groups of women. The first consisted of participants who, at entry into the trial, were infected with 1 or more of 12 oncogenic HPV types (defined herein), and who were randomized, vaccinated, and followed for a period of approximately 4.75 years (median follow-up 56.7 months; interquartile range 52.4−66.5). The second consisted of participants who were randomized and vaccinated, who at some time during follow-up received LEEP treatment for cervical precancer, and who were followed after treatment (median follow-up 27.3 months; interquartile range 15.3−39.8) as part of their participation in the larger trial. This latter group should not be considered formally randomized, because randomization occurred at entry into the main trial and not at the time of LEEP treatment. For both groups of women, we performed analyses at the infection level, as described herein. The trial was reviewed and approved by the US National Cancer Institute and Costa Rica INCIENSA Institute (Instituto Costarriciense de Investigacion y Ensenanza en Nutricion y Salud) human subjects review committees. Participants provided written informed consent.

At enrollment and during the follow-up period, risk factor questionnaires were administered and pelvic examinations performed on sexually experienced women. Specimens were collected at those visits to allow for ThinPrep cytology (Hologic, Marlborough, MA) and HPV DNA testing. Cytology was interpreted via use of the Bethesda system. Women with cytologic evidence of high-grade precancer and those with evidence of persistent low-grade cervical lesions were referred to colposcopy for further evaluation and disease ascertainment. For those referred to colposcopy, biopsies were collected when cervical lesions were evident and treatment by LEEP performed when clinically indicated. Hematoxylin and eosin slides prepared from formalin-fixed and paraffin-embedded materials were used to grade the lesions by the study pathologist in Costa Rica, followed by independent review by a second pathologist in the United States. When grades were discrepant, a third US pathologist blindly evaluated the slides to adjudicate and establish the final diagnosis for analytical purposes.

HPV HC2 testing (Digene Corporation, Silver Spring, MD) with the use of PreservCyt solution (Cytyc Corporation, Marlborough, MA) was performed as per the manufacturer’s instructions on all enrollment (prevaccination) specimens, all specimens collected at the final blinded visit, and to triage equivocal cytology results (atypical cells of undetermined significance) during follow-up. Broad-spectrum polymerase chain reaction−based HPV DNA testing was performed on cells stored in Preservcyt solution at all study timepoints using a previously described procedure based on amplification with the SPF10 primers, a DNA enzyme immunoassay detection of amplimers, and typing using the LiPA line blot detection system (LiPA HPV InnoLiPA HPV genotyping assay SPF10 system, version 1, Labo Bio-medical Products, Rijswijk, the Netherlands). The LiPA detects 25 HPV genotypes, including the 12 oncogenic HPV types of interest for this evaluation (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59). To ensure that HPV-16 and HPV-18 infections were not missed, all specimens that screened positive for HPV DNA via the SPF10 DNA enzyme immunoassay but that were negative for HPV-16 or HPV-18 by LiPA were tested for the presence of HPV-16 and HPV-18 DNA with the use of type-specific primers, as previously described.

A total of 7466 women were enrolled and randomized in our trial. Of these, the 1901 (954 HPV arm; 947 control arm) who tested positive for 1 or more of 12 oncogenic HPV types at randomization were considered for the infection-level analysis that evaluated the fate of HPV infections present at the time of vaccination ( Figure 1 , A). We further excluded from this analysis 1 woman who received a discordant vaccine (0 HPV arm; 1 control arm), 150 women who had evidence of high-grade disease requiring treatment at enrollment (83 HPV arm; 67 control arm), and 39 women with no follow-up time after enrollment (19 HPV arm; 20 control arm).

Study consort diagrams

A, Consort – evaluation of prevalent HPV infections. B, Consort – evaluation of recurrence after LEEP.

HPV , human papillomavirus; HAV , hepatitia A virus; HSIL , high-grade squamous intraepithelial lesion; LEEP , loop electrosurgical excisional procedure.

Hildesheim et al. HPV vaccination after infection and/or treatment. Am J Obstet Gynecol 2016 .

Women included in this analytical cohort (N=1711; 8588 person years of follow-up) had a median of 7 study visits after enrollment (7, interquartile range 5−8 HPV arm; 7, interquartile range 5−8 control arm) and were followed for a median of 56.7 months (56.4, interquartile range 52.0−66.1 HPV arm; 57.0, interquartile range 52.7−66.8 control arm). They had a total of 2434 oncogenic HPV infections at enrollment (1198 HPV arm; 1236 control arm). Compared with women included in our previous publication on this topic, this analysis included an additional 106 women (6.6% increase) and 7136 person years of follow-up (491% increase). Comparison of this analytical cohort by study arm revealed balance with respect to age, lifetime number of partners, cytology results, and HPV status (single vs multiple infections; HPV type) at enrollment ( Table 1 ).

For the infection-level analysis that evaluated recurrence after treatment, we considered 370 women (172 HPV arm; 198 control arm) who were treated with LEEP during the follow-up period ( Figure 1 , B). We further excluded 41 women (21 HPV arm; 20 control arm) who had a LEEP performed but for whom there was no evidence of high-grade disease before the procedure and 18 women (9 HPV arm; 9 control arm) who had no follow-up after treatment. A total of 311 women were therefore included in the analysis (142 HPV arm; 169 control arm). A total of 130 of these women also were included in the analytical cohort of women infected at entry that was described in the preceding paragraph. Women included in this analytical cohort had a median of 3 study visits after treatment (4, interquartile range 3−5 HPV arm; 3, interquartile range 2−5 control arm) and were followed for a median of 27.3 months after treatment (31.8, interquartile range 19.6−39.8 HPV arm; 23.9, interquartile range 11.5-39.8 control arm). These women had a total of 156 oncogenic HPV infections detected after treatment (71 HPV arm; 85 control arm). Comparison of this analytical cohort by study arm revealed balance with respect to age at enrollment ( Table 2 ).

Given the high efficacy of the HPV-16/18 vaccine against new infections and associated lesions, women in the HPV arm for this analytical cohort were relatively more likely to have a LEEP early in the study to treat a condition that was prevalent at enrollment and conversely less likely to have a LEEP later in the study to treat an incident condition arising from an infection initiating after vaccination (23.9% and 39.6% for HPV arm and Control arm had LEEP performed to treat lesions caused by incident infections, respectively). As a result, the median number of months between enrollment and treatment was 28.2 for those in the HPV arm (interquartile range 15.2−44.6 months) and 38.1 for women in the control arm (interquartile range 22.4−50.5 months). We also observed a greater proportion of women in the HPV arm (compared with the control arm) who reported 2+ partners and who had an abnormal cytology at enrollment, and a lower proportion of women in the HPV arm (compared with the control arm) who were HPV DNA negative at enrollment ( Table 2 ).

We chose to use an infection rather than a woman as the unit of analysis because of our interest in the fate of individual HPV infections. A total of 31.4% of infected women had more than 1 HPV type at enrollment. We evaluated the following HPV categories: HPV-16/18 (HPV-16 and/or HPV-18—the primary vaccine types), HPV-31/33/45 (HPV-31 and/or HPV-33 and/or HPV-45—HPV types for which evidence for cross-protection has been documented), and oncogenic types (any of the 12 oncogenic HPV types).

For evaluation of infections and lesions occurring among women infected at entry, we considered the following outcomes: type-specific viral clearance and development of histologically confirmed cervical lesions (CIN1+ and CIN2+). In a subanalysis restricted to infected women with normal cytology, we also considered as an outcome the development of cytologically evident lesions (low-grade squamous intraepithelial lesion or greater and high-grade squamous intraepithelial lesion or greater).

For the evaluation of women treated by LEEP, we considered the following outcomes after LEEP: HPV infection, persistent HPV infection (defined as detection of type-specific HPV at 2 or more consecutive visits after treatment), squamous intraepithelial lesion (SIL), and CIN2+. We also conducted subanalyses that considered outcomes associated with HPV types present and absent before treatment, separately. Sensitivity analyses were performed restricted to women whose LEEP was performed to treat conditions prevalent at enrollment, to ensure that findings were not driven by the differential proportion of incident outcomes observed between study arms. As results from these sensitivity analyses were similar to those observed for our main analyses, they are not presented further.

Vaccine efficacy, a measure of the percent reduction (or increase) in outcome rates observed when the HPV arm is compared against the control arm, was computed, along with the corresponding 95% confidence interval (95% CI) around vaccine efficacy estimates. The generalized estimating equations method was used to account for possible lack of independence between clearance in analysis of more than one infection in the same woman. The estimates of rates of infection that clear from the generalized estimating equations analysis can, therefore, be slightly different from the crude percentages. We present cumulative vaccine efficacy against persistence for several HPV categories. In addition, to cumulative analyses, curves depicting the fate of prevalent infections over time were also developed.

Results

Impact of HPV-16/18 vaccination on infections prevalent at vaccination

We first evaluated whether rates of viral clearance and/or progression to cervical preneoplastic lesions differed by vaccination status. Results are summarized in Table 3 . We found no evidence that HPV-16/18 vaccination altered the fate of an HPV infection present at the time of vaccination. When we evaluated HPV-16/18 infections, for example, vaccine efficacy was −5.4% (95% CI −19, 10) for clearance, −15.5% (95% CI −86, 28) for progression to CIN1+, and 0.3% (95% CI −69, 41) for progression to CIN2+ in the cohort of women with HPV infection but without precancer. A similar lack of vaccination effect was observed when we evaluated infections with HPV-31/33/45 (HPV types for which evidence for partial cross-protection against incident infections and lesions was previously demonstrated) or infections with any oncogenic HPV. These results were consistent when the more restricted cohort of women without cytologic abnormalities at vaccination was evaluated, and among this more restricted cohort when progression to low-grade or high-grade SIL were evaluated. Time-dependent curves depicting the fate of prevalent infections are provided as Supplemental Figures 1 and 2 and, again, do not suggest any measurable effect of vaccination on either clearance or progression of prevalent infections.

Table 3

Impact of HPV-16/18 vaccination on infections prevalent at vaccination

HPV Types	Cohort	Outcome	Arm	No. infections	No. events	No. person-years	Rate per 1000	% vaccine efficacy	95% CI
HPV-16/18	Infected women without precancer	Clearance	HPV	268	243	492	489	−5.4%	[−19, 10]
			Ctrl	313	298	569	517
		CIN1+	HPV	244	32	1201	28.0	−15.5%	[−86, 28]
			Ctrl	295	35	1427	24.2
		CIN2+	HPV	258	25	1301	20.2	0.3%	[−69, 41]
			Ctrl	308	31	1504	20.3
	Infected women without cytologic abnormalities	Clearance	HPV	173	160	326	481	−3.3%	[−19, 16]
			Ctrl	216	208	403	497
		LSIL+	HPV	171	62	642	77.2	−3.5%	[−85, 42]
			Ctrl	216	71	861	74.6
		HSIL+ a	HPV	161	23	744	30.9	−48.6%	[−172, 19]
			Ctrl	200	20	961	20.8
		CIN1+	HPV	171	23	835	28.5	5.5%	[−60, 44]
			Ctrl	214	32	1035	30.2
		CIN2+	HPV	171	17	849	20.7	22.6%	[−40, 57]
			Ctrl	215	29	1050	26.8
HPV-31/33/45	Infected women without precancer	Clearance	HPV	214	206	323	617	4.0%	[−13, 24]
			Ctrl	222	211	348	594
		CIN1+	HPV	201	17	938	18.3	14.0%	[−62, 54]
			Ctrl	210	22	1020	21.3
		CIN2+	HPV	208	14	986	14.4	17.4%	[−65, 59]
			Ctrl	218	19	1070	17.4
	Infected women without cytological abnormalities	Clearance	HPV	144	138	219	617	9.3%	[−12, 35]
			Ctrl	159	151	261	564
		LSIL+	HPV	144	32	578	52.4	−4.2%	[−79, 39]
			Ctrl	158	41	662	50.3
		HSIL+	HPV	144	13	653	20.1	4.2%	[−101, 54]
			Ctrl	158	17	749	21
		CIN1+	HPV	144	14	656	21.5	17.6%	[−63, 58]
			Ctrl	157	20	753	26.1
		CIN2+	HPV	144	12	663	18.2	15.7%	[−76, 60]
			Ctrl	157	17	763	21.6
Oncogenic HPV	Infected women without precancer	Clearance	HPV	1198	1149	1761	599	4.8%	[−3.7, 14]
			Ctrl	1236	1184	1881	571
		CIN1+	HPV	1110	83	5487	14.0	−0.9%	[−42, 28]
			Ctrl	1149	89	5646	13.9
		CIN2+	HPV	1160	62	5817	9.6	3.1%	[−45, 35]
			Ctrl	1208	68	6004	9.9
	Infected women without cytologic abnormalities	Clearance	HPV	788	758	1195	575	1.0%	[−8.8, 12]
			Ctrl	854	822	1311	569
		LSIL+	HPV	779	191	3242	45.2	−10.7%	[−45, 16]
			Ctrl	847	197	3615	40.8
		HSIL+	HPV	779	64	3750	14.3	−19.2%	[−84, 23]
			Ctrl	847	62	4128	12
		CIN1+	HPV	779	62	3814	15.5	6.7%	[−35, 35]
			Ctrl	842	76	4130	16.6
		CIN2+	HPV	779	46	3862	10.8	15.0%	[−31, 45]
			Ctrl	845	59	4188	12.8

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