Abstract
Background
The link between squamous cell cervical carcinoma and HPV 16/18 is well-established but the magnitude of the risk association is uncertain and the importance of other high-risk HPV types unclear.
Methods
In two prospective nested case-control series among women participating in cytological screening in Sweden, we collected 2772 cervical smears from 515 women with cancer in situ (CIS), 315 with invasive squamous cell carcinoma (SCC), and individually matched controls. All smears were tested for HPV with PCR assays and the median follow-up until diagnosis was 5-7 years. Conditional logistic regression was used to estimate relative risks (RR) and 95% confidence intervals (CI).
Results
Presence of HPV16/18 in the first smear was associated with 8.5-fold (95% CI 5.3-13.7), and 18.6-fold (95% CI 9.0-38.9) increased risks of CIS and SCC, respectively, compared to women negative for HPV. Infection with other high-risk HPV types in the first smear was also associated with significantly increased risks for both CIS and SCC. Persistence of HPV16 infection conferred a RR of 18.5 (95% CI, 6.5-52.9) for CIS and 19.5 (95% CI 4.7-81.7) for SCC. The HPV16/18 attributable risk proportion was estimated to 30-50% of CIS, and 41-47% of SCC. Other high-risk HPV types also conferred significant proportions.
Conclusions
Our large population-based study provides quantification of risks for different HPV types and prospective evidence that non-16/18 high-risk HPV types increase the risk for future cervical cancer.
Impact
This study gives further insights into cervical cancer risk stratification with implications for HPV-based prevention strategies.
Keywords: Cervical cancer, HPV, risk, prevalence, persistence
Introduction
With new vaccines against the two most oncogenic human papillomavirus types (HPV16 and HPV18), the future incidence of cervical cancer is likely to radically decrease. Randomized trials have shown virtually complete protection against HPV16 and HPV18-related CIN2+ following prophylactic HPV vaccination (1-3). Nevertheless, it will take decades for vaccination to take effect on cervical cancer rates and the need to improve screening practices continues. Uncertainties also exist regarding the future dynamics of non-16/18 high-risk HPV types following vaccine deployment, including the degree and duration of cross-protection of the current vaccines against these other types. For women currently undergoing cervical screening, HPV-based screening has emerged as the way to improve prevention (4, 5). Hence, close investigation of the future cancer risks associated with HPV infection of different types – one of the world's most common sexually transmitted infections – is needed in order to develop sensible tools for risk stratification. To this end, we prospectively examined the risks of developing in situ and invasive squamous cell cervical carcinoma associated with different HPV types and with persistent HPV16 infection, in a large population-based cohort of Swedish women. Our results may carry implications for future HPV-based screening programs, and continued development of HPV vaccines.
Methods
Participants
Starting in 1967, cytological screening with Papanicolaou (Pap) smears was gradually introduced in Sweden. Since the mid-1970's, all Swedish women have been invited for screening every 3 or 4 years (6). Virtually all smears have been stored, and records containing all information from the cytological screening are computerized in the Swedish National Cervical Screening Register (NCSR) (7). The Swedish National Cancer Registry (NCR), established in 1958, records all new diagnoses of both cancer in situ and invasive cervical cancer. The register is considered to be virtually 100% complete (8).
The source population for this study comprised all Swedish women (757690) who participated in cervical screening within at least one of seven Swedish counties sometime during the period 1969-2002. Using the NCSR, we identified a cohort of 739072 women whose first registered smear during the study period was classified as cytologically normal (PAP=1). Records from our cohort were then linked to the NCR to identify a random sample of women with a first diagnosis of cancer in situ (CIS), and all women with a first diagnosis of invasive squamous cell carcinoma (SCC) after entry in our study. A diagnosis of CIS in the Swedish NCR translates to a diagnosis of cervical intraepithelial neoplasia, grade 3 (CIN3).
We identified 515 CIS cases, and 315 SCC cases. Using case-control sampling, one woman – matched on county, date of entry into cohort (+/- 3 months), and age at first normal smear (+/- 1 year) – was randomly selected as an individually matched control for each CIS and SCC case. The smears taken prior to the date of diagnosis of the case in each case-control pair were identified and requested from the archives. To verify the diagnoses of CIS or SCC, histological specimens from the identified cases were reviewed by our pathologist (AL).
Smear Analyses
Each smear was re-coded and re-labelled to ensure blinding of case-control status during DNA extraction and HPV analysis. Samples belonging to the same case-control pair were included in the same analysis batch. DNA extraction was performed by validated methods described elsewhere (9). The risk for cross-contamination of archival smears has been evaluated and found to be low (9). All smears were analysed for the presence of seven low-risk HPV types (HPV 6, 7, 11, 42, 43, 70, and 90), and 16 high-risk HPV types (HPV16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68, 73, and 82). The polymerase chain reaction (PCR) amplification of a consensus region using GP5+/6+ primers (10), was followed by detection of biotinylated HPV amplicons by hybridisation to short oligonucleotide probes covalently linked to fluorescence-labeled carboxy-coated polystyrene beads on the Bioplex 200 Luminex system (Biorad, CA, USA), as described previously (11). Positive controls (HPV16 DNA) and multiple negative controls (Sigma water) were included in all runs to ensure absence of contamination. The amount of amplifiable DNA in the samples was determined by real-time PCR for the housekeeping β-globin gene. All laboratory analyses were performed with the analysing laboratory (the WHO HPV LabNet Global Reference Laboratory, Malmö, Sweden) being blinded to information about the samples.
Initially, 4526 smears were retrieved from the archives. We subsequently excluded 102 smears with negative β-globin value and 18 with inconclusive HPV status. Furthermore, we excluded 50 smears from 23 incomplete case-control pairs – because either the case or the control did not have any eligible Pap smears – and 20 smears that were taken on the day of the case's diagnosis.
For the analysis of risk associations with different HPV types, we present results from the first and last smear, comprising a total of 2772 smears from 830 (515 CIS and 315 SCC) complete case-control pairs. Persistence of HPV16 infection was analysed using the first two consecutive smears (hence women with only one smear were excluded). Cases with both smears taken within the year of their diagnosis were excluded (only applicable to one case-control pair). Using these criteria, 1584 smears from 396 (241 CIS and 155 SCC) complete case-control pairs remained for the persistence analysis.
Statistical Analyses
Due to the matched design, conditional logistic regression was used to estimate odds ratios (ORs). These were interpreted as estimates of relative risk (RR) of CIS or SCC in HPV exposed women, relative to women unexposed to the HPV strain/-s under investigation; and relative to women unexposed to any HPV. Pooled risk estimates were calculated for all low-risk HPV types (LRHPV) and for all high-risk HPV types excluding HPV16 and 18 (non-16/18 HRHPV). We analyzed separately the risk associated with HPV presence in the first, and in the last smear prior to the case's diagnosis.
We defined exposure categories as follows; a) HPV16 - the first/last smear being positive for HPV16; b) HPV18 - the first/last smear being positive for HPV18; c) HPV16/18 - the first/last smear being positive for HPV16 and/or 18; d) non-16/18 HRHPV - the first/last smear being positive for one or more high-risk HPV types but not HPV16 or 18; e) LRHPV - the first/last smear being positive for one or more low-risk HPV types.
Persistence of HPV16 infection in the first two consecutive smears was defined as: a) negative (both smears negative for HPV16); b) transient (first positive, second negative); c) acquired (first negative, second positive); d) persistent (both smears positive for HPV16).
Attributable risk proportions and 95% confidence intervals (CI) were calculated based on ORs obtained from the conditional logistic regression models (12). All odds ratios were estimated using STATA version 10 (College Station, TX: Stata Corp.).
The study was approved by the Karolinska Institutet Ethics Review Board, which also determined that informed consent from the participants was not required.
Results
Characteristics of the Participants
Characteristics of the 515 CIS and 315 SCC cases and their matched control women are described in Table 1. The CIS cases were younger at time of diagnosis (median age 33, range 18-64 years), compared to the SCC cases (median age 40, range 24-81 years). The time elapsed from the last smear to date of diagnosis was 0.6 years for CIS cases, giving an average interval of 4.7 years between the first and last smear. For CIS controls, this interval was around 3 years. The study time from the first smear for SCC cases and controls was around 7 years (range 0.01-17.8 years). The time elapsed from the last smear to date of diagnosis was 1.8 years for SCC cases and 2.8 years for their controls. This corresponded to an interval of around 5.5 years between the first and last smear for cases and just under 5 years for controls. The number of smears registered during follow-up was fairly similar for all women, with a median of 2 (range 1- 15). In the persistence analysis, the median time interval between the first and the second smear was 3 years (range 0.13-11.6 years), which is the recommended screening interval in Sweden. This interval was very similar in the different exposure categories (negative, transient, acquired or persistent).
Table 1.
Characteristics of the participants. Median age and study time in years (range).
| Cancer in situ | Invasive squamous cell carcinoma | |||
|---|---|---|---|---|
| Cases | Controls | Cases | Controls | |
| Number of study subjects | 515 | 515 | 315 | 315 |
| Age at diagnosis1 | 33 (18-64) | 33 (18-63) | 40 (24-81) | 40 (24-81) |
| Age at entry | 26 (15-60) | 26 (15-60) | 32 (15-78) | 32 (15-75) |
| Study time – time from first smear to diagnosis | 5.3 (0.03-17.5) | 5.4 (0.01-17.3) | 7.2 (0.01-17.8) | 7.6 (0.06-17.8) |
| Time from last smear to diagnosis | 0.6 (0.01-16.2) | 2.4 (0.01-16.2) | 1.8 (0.01-15.9) | 2.8 (0.01-14.7) |
| Median no. of smears per subject (range) | 2(1-13) | 2(1-11) | 2(1-15) | 2(1-11) |
For controls age at time of diagnosis of their respective case
HPV prevalences
The prevalence of any HPV in the first smear at study entry was tripled in CIS and SCC cases, compared to controls (59% and 61% versus 18%, respectively, Table 2). Most of the infections were HPV16/18 in both CIS cases (35%) and SCC cases (44%). Multiple HPV types were present in the first smear of 11% of CIS cases and 9% of SCC cases, but only in 3-4% of controls. Table 2 also gives HPV prevalences in the last smear preceding diagnosis of CIS or SCC. The HPV prevalence increased from first to last smear in both CIS and SCC cases, whereas the prevalence among controls diminished slightly.
Table 2.
Prevalence of specific HPV types and HPV type groupings in first and last Pap smear for cancer in situ (CIS), and invasive squamous cell carcinoma (SCC) cases and their matched controls. HRHPV denotes High-Risk HPV and LRHPV Low-Risk HPV.
| Cancer in situ | Invasive squamous cell carcinoma | ||||||||
|---|---|---|---|---|---|---|---|---|---|
| First smear | Last smear | First smear | Last smear | ||||||
| HPV Type | High-risk (HR) or Low-risk (LR) | Cases N=515 n (%) * | Controls N=515 n (%) * | Cases N=515 n (%) * | Controls N=515 n (%) * | Cases N=315 n (%) * | Controls N=315 n (%) * | Cases N=315 n (%) * | Controls N=315 n (%) * |
| Any HPV | HR/LR | 301 (59) | 94 (18) | 407 (79) | 72 (14) | 191 (61) | 58 (18) | 224 (71) | 45 (14) |
| HPV-16/18 | HR | 179 (35) | 34 (7) | 262 (51) | 26 (5) | 140 (44) | 21 (7) | 158 (50) | 17 (5) |
| Non-16/18 HRHPV | HR | 146 (28) | 54 (11) | 198 (39) | 35 (7) | 65 (21) | 35 (11) | 77 (24) | 20 (6) |
| Any HRHPV | HR | 288 (56) | 82 (16) | 396 (78) | 57 (11) | 184 (58) | 49 (16) | 220 (70) | 33 (11) |
| Any LRHPV | LR | 34 (7) | 19 (4) | 32 (6) | 16 (3) | 12 (4) | 13 (4) | 8 (3) | 12 (4) |
| Multiple HPV types | HR/LR | 57 (11) | 14 (3) | 82 (16) | 9 (2) | 28 (9) | 11 (4) | 19 (6) | 6 (2) |
| HPV-6 | LR | 7 (1) | 2 (0) | 4 (0) | 3 (1) | 1 (0) | 0 | 0 | 2 (1) |
| HPV-7 | LR | 0 | 0 | 1 (0) | 1 (0) | 0 | 0 | 0 | 0 |
| HPV-11 | LR | 5 (1) | 1 (0) | 4 (0) | 1 (0) | 0 | 1 (0) | 0 | 1 (0) |
| HPV-16 | HR | 158 (31) | 28 (5) | 235 (46) | 19 (4) | 118 (38) | 16 (5) | 131 (42) | 12 (4) |
| HPV-18 | HR | 30 (6) | 7 (1) | 36 (7) | 8 (2) | 27 (9) | 6 (2) | 29 (9) | 6 (2) |
| HPV-31 | HR | 34 (7) | 17 (3) | 49 (10) | 8 (2) | 13 (4) | 7 (2) | 16 (5) | 3 (1) |
| HPV-33 | HR | 34 (7) | 5 (1) | 41 (8) | 4 (1) | 16 (5) | 2 (1) | 16 (5) | 2 (1) |
| HPV-35 | HR | 11 (2) | 3 (1) | 14 (3) | 2 (0) | 2 (1) | 4 (1) | 3 (1) | 0 |
| HPV-39 | HR | 5 (1) | 3 (1) | 7 (1) | 4 (1) | 4 (1) | 1 (0) | 5 (2) | 1 (0) |
| HPV-42 | LR | 16 (3) | 12 (2) | 20 (4) | 8 (2) | 7 (2) | 9 (3) | 6 (2) | 7 (2) |
| HPV-43 | LR | 3 (1) | 2 (0) | 1 (0) | 1 (0) | 1 (0) | 2 (1) | 1 (0) | 1 (0) |
| HPV-45 | HR | 16 (3) | 3 (1) | 24 (5) | 4 (1) | 15 (5) | 6 (2) | 25 (8) | 7 (2) |
| HPV-51 | HR | 8 (2) | 5 (1) | 14 (3) | 3 (1) | 3 (1) | 2 (1) | 1 (0) | 1 (0) |
| HPV-52 | HR | 18 (4) | 4 (1) | 24 (5) | 5 (1) | 3 (1) | 4 (1) | 4 (1) | 1 (0) |
| HPV-56 | HR | 14 (3) | 9 (2) | 19 (4) | 8 (2) | 5 (2) | 2 (1) | 9 (3) | 2 (1) |
| HPV-58 | HR | 10 (2) | 3 (1) | 15 (3) | 3 (1) | 1 (0) | 2 (1) | 1 (0) | 2 (1) |
| HPV-59 | HR | 26 (5) | 10 (2) | 33 (6) | 7 (1) | 15 (5) | 9 (3) | 13 (4) | 6 (2) |
| HPV-66 | HR | 10 (2) | 5 (1) | 13 (3) | 3 (1) | 5 (2) | 5 (2) | 3 (1) | 4 (1) |
| HPV-68 | HR | 1 (0) | 1 (0) | 2 (0) | 1 (0) | 1 (0) | 1 (0) | 2 (1) | 0 |
| HPV-70 | LR | 4 (1) | 3 (1) | 2 (0) | 4 (1) | 3 (1) | 2 (1) | 1 (0) | 2 (1) |
| HPV-73 | HR | 0 | 1 (0) | 0 | 2 (0) | 1 (0) | 2 (1) | 0 | 1 (0) |
| HPV-82 | HR | 1 (0) | 0 | 3 (1) | 0 | 0 | 0 | 0 | 0 |
Addition of individual HPV types exceeds the number of women due to multiple HPV infections in some women. Therefore, the sum of percentages of positive HPV infections may exceed 100%.
Among both CIS and SCC cases, the probability of being positive for HPV16/18 at 15 years before diagnosis was around 20% (Figure 1a). The probability increased as time to diagnosis decreased, to over 40% for CIS cases, and 60% for SCC cases one year prior to diagnosis (Figure 1a). The probability for a non-16/18 HRHPV infection was 10% 15 years prior to, and increased to nearly 40% at time of CIS diagnosis. For SCC cases, this probability remained around 20% from 15 years before to time of diagnosis. For controls, the probability remained stable over time with around 5-10% infected with HPV16/18 or other high-risk types (Figures 1a, 1b). The probability of being infected with LRHPV was similar for CIS/SCC cases and controls, and remained constantly low over time (Figure 1c).
Figure 1.
A-C: Proportion of HPV infected (first smear only) women by cancer type, HPV subtype and case/control status (the size of the scatters is proportional to the number of women that were included in the calculations).
Risk Associations with HPV type
The relative risks increased considerably when using a reference group negative for HPV, as opposed to using a mixed reference group (Table 3). This was particularly evident in the last smear. Being positive for HPV16/18 in the first smear conferred an almost 9-fold (RR 8.5, 95% CI 5.3-13.7) increased risk for CIS, and 19-fold (RR 18.6, 95% CI 9.0-38.9) increased risk for SCC, compared to being HPV negative. These risk associations increased in the last smear, with a more pronounced increase for CIS than for SCC.
Table 3.
Conditional logistic regression showing odds ratios (OR) and 95% confidence interval (CI) of cancer in situ (CIS) and invasive squamous cell carcinoma (SCC) in the first and last smears among HPV positive women. HRHPV denotes High-Risk HPV and LRHPV Low-Risk HPV. Non-16/18 HRHPV denotes any high-risk HPV infection apart from HPV16 or HPV18.
| Reference: All other womenΨ | Reference: HPV negative womenΨ,† | |||
|---|---|---|---|---|
| RR (95% CI) | RR (95%CI) | |||
| First smear | Last smear | First smear | Last smear | |
| Cancer in situ | ||||
| HPV16/18 | 6.2 (4.2-9.2) | 22.5 (12.3-41.1) | 8.5 (5.3-13.7) | 37.1 (18.1-76.1) |
| Non-16/18 | ||||
| HRHPV | 3.5 (2.4-5.0) | 8.4 (5.4-13.1) | 4.4 (2.8-6.8) | 15.8 (8.3-30.1) |
| LRHPV | 1.8 (1.0-3.1) | 2.0 (1.1-3.7) | 2.6 (0.97-6.8) | 5.0 (1.4-17.9) |
| Squamous cell carcinoma | ||||
| HPV16/18 | 12.9 (6.8-24.6) | 15.1 (8.0-28.6) | 18.6 (9.0-38.9) | 28.5 (13.0-62.4) |
| Non-16/18 | ||||
| HRHPV | 2.0 (1.3-3.1) | 4.6 (2.7-7.8) | 3.0 (1.6-5.5) | 16.9 (6.8-42.1) |
| LRHPV | 0.9 (0.4-2.1) | 0.7 (0.3-1.6) | 1.3 (0.4-4.5) | 0.8 (0.1-4.8) |
All estimates were controlled for matching criteria (county, date of entry into cohort, and age at first normal smear).
RR's further adjusted for all other HPV variables.
The risk of developing CIS or SCC for women infected with non-16/18 HRHPV in the first smear was increased both for CIS (RR 4.4, 95% CI, 2.8-6.8) and for SCC (RR 3.0, 95% CI 1.6-5.5), compared to HPV negative women (Table 3). The risk associations were increased in the last smear, with RR's of 15.8 (95% CI 8.3-30.1) for developing CIS, and 16.9 (95% CI 6.8-42.1) for developing SCC. Due to a lack of statistical power for HPV types of low prevalence, we could not obtain reliable estimates for the individual non-16/18 HRHPV types.
Separate analyses for HPV16 and 18 were performed using a combined endpoint of CIS and SCC. Women exposed to HPV16 in their first smear had approximately the same risk elevation as those exposed to HPV18 (RR's 11.0, 95% CI 7.2-16.8; vs 12.2, 95% CI 4.4-34.1, respectively), compared to HPV negative women. The risk estimation for HPV16 in the last smear increased substantially (RR 41.4, 95% CI 22.7-75.5), whereas the risk for HPV18 increased only modestly (RR 16.2, 96% CI 6.1-43.0) (data not shown).
HPV type persistence
Among cases, 35% (84/241) of CIS and 34% (53/155) of SCC were positive for the same HPV type in the first and second smear, compared to only 4% (9/241 and 6/155, respectively) of the controls. The majority of persistent infections among both cases and controls were attributable to HPV16 (Figure 2). Compared to women negative for HPV 16 in the first and second smear, being transient conferred no increased risk of CIS, and a non-significantly increased risk of SCC, while having acquired a HPV infection conferred increased risks of both CIS and SCC. Persistence of HPV16 showed a 19-fold increased risk for CIS (RR 18.5, 95% CI 6.5-52.9) and a 20-fold increased risk for SCC (RR 19.5, 95% CI 4.7-81.7) (Table 4).
Figure 2.
Proportions (%) of women with persistent infections due to different HPV types among 241 cancer in situ (CIS) and 155 squamous cell carcinoma (SCC) case women and their matched controls. 25% of CIS and SCC cases (60/241 and 38/155 women, respectively) were persistent for HPV 16.
Table 4.
Conditional logistic regression showing odds ratios (OR) and 95% confidence interval (CI) of cancer in situ (CIS) and invasive squamous cell carcinoma (SCC) among women with transient, acquired, or persistent HPV16 infection compared to those negative for HPV16 in both the first and second smear.
| Conditional analysis‡ | CIS (Ncases = 241 Nctrls = 241) |
SCC (Ncases = 155 Nctrls = 155) |
||
|---|---|---|---|---|
| OR | 95 % CI | OR | 95 % CI | |
| HPV16 status in the first and second smear | ||||
| Negative -/- | 1.00 | Reference* | 1.00 | Reference* |
| Transience +/- | 1.2 | 0.5-3.1 | 2.6 | 0.8-8.2 |
| Acquisition -/+ | 7.6 | 2.8-20.5 | 2.9 | 1.2-7.3 |
| Persistence +/+ | 18.5 | 6.5-52.9 | 19.5 | 4.7 – 81.7 |
Estimates were controlled for matching criteria (county, date of entry into cohort, and age at first normal smear).
Attributable risk proportions
In the studied cohort, the HPV16/18 attributable risk proportions (ARP) in the first smear were 29% (95% CI 24-34%) for CIS, and 41% (95% CI 35-47%) for SCC. The corresponding HPV16/18 ARP's in the last smear were 49% (95% CI 44-53%) for CIS, and 47% (95% CI 41-53%) for SCC. In the first smear, 20% (95% CI 15-25%) of CIS cases, and 10% (95% CI 4-16%) of SCC cases, were attributable to non-16/18 HRHPV types. These ARP's increased to 34% (95% CI 29-39%) of CIS cases, and 19% (95% CI 13-25%) of SCC cases in the last smear.
Discussion
This large population-based study prospectively investigated the risk of developing in situ and invasive squamous cervical cancer (CIS and SCC) in relation to the presence of all major genital HPV types. It also covers virtually the whole time period of active cervical screening in Sweden. Women with a cytologically normal smear but infected with HPV16 and/or HPV18 already more than 15 years before diagnosis, exhibited a much higher risk for SCC than HPV negative women. Our results further suggest that infection with other high-risk HPV types also carry significant risks, which may be relevant in the context of HPV vaccination and HPV-based screening.
Much has now been described of the natural history of HPV infection, and its causal link to cervical cancer. It is also well-known to be a common infection that will progress to more severe disease in relatively few women (13). The challenge remains to disentangle which women are at future clinical risk, and identify robust markers of disease progression (14). Although the knowledge base in this area has increased considerably, there have been some limitations to previous studies that have investigated the risks for CIS or SCC imposed by the common oncogenic HPV types. Some studies lack a population-based structure (15, 16) and/or are limited to single municipalities (15, 17-20). Additionally, most studies have limited their HPV typing to one or a few HPV types (15, 17, 20-22) and/or have only assessed HPV types using serological assays (21, 23). Other prospective population-based studies have included only one outcome (18, 24). Our study significantly advances the prior knowledge because of our large sample size and complete HPV typing that estimated risks associated with the different HPV types for both CIS and SCC within the same population-based cohort comprising all age groups. However, since this study analyzed HPV-status only in women who participate in screening – which the large majority of Swedish women do – we cannot draw inference regarding women who do not. And although they are population-based, control women were matched to case women on certain criteria and may therefore not be totally representative of the general female population.
While a meta-analysis recently validated the use of 6-12-month persistence of any HPV infection as a clinical risk marker, the authors noted that few studies in the analysis focused on risks associated with type-specific persistence (25). Furthermore, it is unclear how many cases of invasive cancer were included in the study's combined endpoint (CIN2-3/HSIL+). Other studies have arrived at the same conclusion but have, as yet, only assessed small numbers of CIN1+ or CIN2+ (26-28).Whereas we concur with these findings, we were able to substantiate the concept of persistence also in a large cohort of higher-grade/invasive disease cases. In our study, we observed that being positive for HPV16 in two consecutive smears resulted in close to 17- and 20-fold increased risks for CIS and SCC, respectively, compared to women negative for HPV16 in both smears. The risk was also much higher than that for transiently infected women, or women with a new infection. These estimates demonstrate that women with persistent infection with HPV16 are at a substantially increased risk compared to all other women, HPV infected or not. HPV persistence is increasingly being used as an intermediate endpoint of invasive cervical cancer, both in HPV vaccination trials and in cervical screening strategies. However, this strategy is not yet universally accepted. Our study provides prospective evidence directly supporting the idea that HPV persistence is indeed an important event in the pathway of cervical carcinogenesis.
It should be noted at this point that although our study design allows us to prospectively study cervical disease, it cannot distinguish between persistent or recurrent infection. Since the time interval between two smears in this study was on average 3 years, we acknowledge that some of our persistent infections may be re-infections with the same type, although many participants in our study were over the age of 30 and thus past the peak in HPV infection incidence. Due to the time interval between smears, we also emphasize that these findings should not be interpreted in a strict natural history sense, and it was not the aim of this study to describe such a context.
The risk estimates conferred by other (non-16/18) high-risk HPV types in the first smear were lower for SCC, compared to the risk associated with having a first smear positive for HPV16/18. However, non-16/18 HRHPV types in the last smear imposed considerably higher risk for future CIS, and even SCC development, compared to HPV negativity. Hence, the role of these HPV infections may be important. Indeed, one study found a higher risk of developing CIN2+ among HPV31 and HPV33 positive women than HPV18 positive women (relative to HPV negative women) (24). The added risk for cervical cancer conveyed by non-16/18 HRHPV types has direct implications for the efficacy of HPV vaccines. Whereas some evidence exists that a certain degree of cross-protection can occur with the current HPV vaccines (1, 3, 29), this appears to be limited to those HPV's that are phylogenetically related to HPV16 and 18, particularly HPV31, HPV45 and HPV52 (30). It is therefore likely that current vaccines will only partially protect against oncogenic HPV types other than HPV16/18.
We estimate that 41-47% of SCC cases in this cohort were attributable to HPV16/18, and thus potentially preventable through current HPV vaccines. The corresponding figure for CIS we estimate to 30-50%, which is in line with a previous study that found a 39% reduction in CIN2+ by removal of HPV16/18 from the Swedish population (24). Furthermore, we found that other remaining high-risk types accounted for at least 10-20% of all SCC in our cohort, which illustrates the benefit that could be derived from cross-protection or future expansion with inclusion of additional HPV types in the vaccines. Our ARP's may be overestimates due to issues of population coverage, compliance, and a lower likely efficacy of the vaccine in general population than in clinical studies. In contrast, our reliance on women's cervical smears rather than their diagnostic samples, and/or lower sensitivity in analyses based on archival smears, could mean that we under-estimated the true values. Extensive typing of tumor tissue has demonstrated near 100% prevalence of HPV DNA in squamous cervical lesions, 70% of which have been attributed to HPV16/18 (13, 31). In the analysis of smears from our case women, around 20% remain HPV negative even in the last smear before diagnosis. Since HPV is recognized as a necessary factor for squamous cervical cancer development (32), and all known HPV types were tested for, we conclude that our assay has around 80% sensitivity for archival smear HPV typing. This sensitivity did not vary according to how long the smears had been stored in the archive (data not shown). Despite the somewhat limited sensitivity, we still consider our estimates of relative contributions of different HPV types to be of value since they offer a complementary perspective to that derived from tumor tissue, albeit a conservative such.
In summary, our population-based study provides quantification of HPV-type-specific and HPV-persistence-specific cervical cancer risks. We also provide prospective evidence for a strong role of non-16/18 high-risk HPV types in both CIS and SCC development. The ability for current vaccines to exhibit cross-protective properties would no doubt show considerable benefit to the community, as would the introduction of a further vaccine(s) against other high-risk HPV types.
Acknowledgments
Ninoa Malki for database management and Aline Marshall, Carina Eklund and Kia Sjölin in the Joakim Dillner lab for the HPV testing.
FUNDING
This work was supported by the National Cancer Institute at the National Institutes of Health (grant numbers 1R01CA093378-01A1, 5R01CA111720-03). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Cancer Institute or the National Institutes of Health.
Footnotes
CONFLICT OF INTEREST STATEMENT
Pär Sparén reports consulting and board membership for GlaxoSmithKline and receiving grants from SanofiPasteur Sweden.
Joakim Dillner reports consulting and board membership for Merck and Co., Inc. and receiving grants from Merck and Co., Inc. and SanofiPasteur MSD. He also reports receiving lecture honoraria from SanofiPasteur MSD. He has also received reimbursement for travel expenses from Merck and Co., Inc., and reimbursement for grant review from GlaxoSmithKline.
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