Epidemics 2 (2010) 2128

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Epidemics
j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / e p i d e m i c s

HPV-16 infection and cervical cancer: Modeling the inuence of duration of infection and precancerous lesions
Iacopo Baussano a,b,,1, Guglielmo Ronco c, Nereo Segnan c, Katherine French a, Paolo Vineis d, Geoff P. Garnett a
a

Department of Infectious Disease Epidemiology, St. Mary's Campus, Imperial College, London W2 1PG, UK SCDU Epidemiologia dei Tumori, CPO-Piemonte, Novara - 28100, Italy Epidemiologia dei Tumori, CPO-Piemonte, Torino - 10100, Italy d Department of Epidemiology and Public Health, St. Mary' Campus, Imperial College, London W2 1PG, UK
b c

a r t i c l e

i n f o

a b s t r a c t
The patterns of transmission, clearance, and progression of HPV infection and the related precancerous lesions are key to accurately model cervical cancer epidemiology and prevention. We have developed an age-structured dynamic model of the transmission of HPV-16 infection. This mathematical model accounts, for the rst time, for the effect of infection and precancerous lesions duration on the natural history of HPV-16 infection and precancerous lesions. The model's output has been tted to contemporaneous sets of data from Turin, Italy, to estimate parameters that have had been indirectly tested by comparing them with other estimates reported in the literature. The average probability of HPV-16 infection transmission per sexual partnership was about 40%. The HPV-16 clearance and progression rates decreased as the length of time with infection increased, clearance ranging between 1.6 per woman-year (in the rst 6 months of infection) and 0.036 (after more than 6 years of infection), and progression between 0.072 and 0.018 per woman-year. The rate of clearance of precancerous lesions (CIN2+) was inversely dependent on age, while the progression of CIN2+ toward invasive cervical cancer increased as the precancerous lesions persisted. The present study also suggests that an exclusive role of women's age in shaping the rate of progression to cancer is unlikely. These results should inform future analyses. Including more accurately the role of the duration of infection and precancerous lesions as determinants of the cervical cancer occurrence in models of cervical cancer control may inuence predictors of the effectiveness of intervention strategies. 2010 Elsevier B.V. All rights reserved.

Article history: Received 30 November 2009 Revised 29 January 2010 Accepted 2 February 2010 Keywords: Human papillomavirus Cervical cancer Screening Mathematical modeling

Introduction Infection with the carcinogenic types of human papillomaviruses (HPVs) is one of the most important identied risk factors for human cancer (Bosch et al., 2002). Some 15 genotypes are known to put women at high risk (HR) of developing cervical cancer. HPV-16 and HPV-18 are responsible for about 50% of cervical intraepithelial neoplasia (CIN) grade 3 and about 70% of the half a million new cases of cervical cancer occurring worldwide every year (Parkin et al., 2005; Smith et al., 2007). HPV-16 and -18 are also the viral types with the
Abbreviations: HPV, human papillomavirus; SIR, susceptible infected recovered; SIS, susceptible infected susceptible; CIN, cervical intraepithelial neoplasia. Corresponding author. Imperial College of Science, Technology and Medicine, Division of Epidemiology, Public Health and Primary Care, Faculty of Medicine, room 512 (fth oor), St Mary's Campus, Norfolk Place, London W2 1PG, UK. Fax: + 44 20 74022150. E-mail address: i.baussano06@imperial.ac.uk (I. Baussano). 1 SCDU di Epidemiologia dei Tumori, Servizio di Statistica Medica, CPO Piemonte, Via Solaroli, 17, Novara - 28100, Italy. Fax: +39 0321 3733112. 1755-4365/$ see front matter 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.epidem.2010.02.002

strongest oncogenic potential with respect to the other carcinogenic types. Khan et al. (2005) have shown, following a cohort of more than 20,000 women, that the 10-year cumulative incidence rate of CIN3 or cancer was 17% among women who tested positive for HPV-16 at enrolment, while it was 14% and 3% among women who tested positive for HPV-18 and other carcinogenic HPV types, respectively. The incubation period from the infection with high-risk HRHPV types, peaking in women younger than 25 years (de Sanjose et al., 2007) of age and the occurrence of invasive cervical cancer, peaking in women older than 40 years (Gustafsson et al., 1997) of age, is highly variable and can last decades. The biological mechanisms underlying progression and clearance of infection and precancerous lesions are still poorly understood (Woodman et al., 2007), while observational data on the evolution of precancerous lesions are exceptionally rare, given that as part of clinical management they should be removed when identied. The combined effect of these biological mechanisms, separately assessed in different studies, can be represented by accounting for the women's age, the duration of infection (dened as time elapsed since infection)

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and the duration of the precancerous lesions (dened as time elapsed since precancerous lesions occurrence) in a mathematical model. The characterization of the patterns of clearance and progression of infection and precancerous lesions is essential to accurately model the impact of interventions such as screening and vaccination. In particular, to dene the most appropriate age ranges and time intervals to combine screening and vaccination, it is necessary to account for the rate of occurrence, clearance, and progression of HPV infection and precancerous lesions and their determinants. To investigate the natural history of HPV-16 infection and occurrence of related cervical cancer, under the hypothesis that the duration of infection and precancerous lesions (CIN2+) determine the patterns of progression and clearance at each stage of the development of cervical cancer, we developed and analysed an agestructured dynamic model (Fig. 1). Through comparing model outputs to multiple stages of disease detected in surveys and routine healthcare, we investigated qualitatively and quantitatively the relative importance of women's age, assumed as a key determinant of progression toward cervical cancer in previously published models, compared to the duration that a lesion has persisted (lesion's age) in determining the chance of cancer occurrence. Materials and methods Model structure We developed an age-structured model accounting for the effect of duration of infection and of precancerous lesions on the natural

history of the cervical cancer. This model, representing the dynamics of the HPV-16 infection and cervical cancer natural history (Fig. 1), stems from descriptions published by Trottier and Franco (2006) and by Barnabas et al. (2006). Our model accounts only for HPV-16 infection and consequently only for the HPV-16-related precancerous lesions and cervical cancers. Thus, the rates of clearance and progression of infection and precancerous lesions presented in this article refers exclusively to HPV-16 infection and related lesions. Rates of clearance and progression of HPV-16 infection and CIN2+ lesions are allowed to change according to the women's age and the duration of infection itself and the duration of the lesions associated with HPV-16 infection to explore their effect on the patterns of cervical cancer occurrence. The model's outputs were calibrated against contemporaneous sets of data on the occurrence of HPV-16 infection, of CIN2+ lesions, and of invasive cervical cancers observed, between 1998 and 2006, in Turin (population 900,608), Italy. The population has been subdivided into susceptible, HPV-16infected, immune to HPV-16, with CIN2+ lesions, with precancerous lesions detected and treated through screening and with invasive cervical cancer. Furthermore, the population has been stratied, by age groups and into three classes of sexual activity (CSA), according to the average number of new sexual partners per year as reported in the third Italian national survey on knowledge, attitudes, and sexual behavior in relation to HIV/AIDS risk from 2002 (Signorelli et al., 2006). The model is a system of partial integro-differential equations solved numerically in the C programming language. The full mathematical description of the model and the parameterization details are reported in the electronic Supplementary data.

Fig. 1. HPV-16 transmission model. The dynamics of the infection transmission and occurrence of cervical lesions account for calendar time, women's age, and time elapsed since infection (or infection duration) and time elapsed since precancerous lesions occurrence (or lesion duration). Note that the dimensions of the arrows connecting the different compartments are not proportional to the rates of transition. For the model's details, see Supplementary data.

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Demography, sexual behavior, and secondary prevention Some demographic, behavioral, and prevention parameters of the model were xed, namely variations over calendar time of age-specic fertility rates, age- and gender-specic mortality rates, the sexual contact matrix, and the performance of the local screening program. Demographic rates were obtained from the National Institute of Statistics (ISTAT); the sexual mixing matrix was dened using data collected in the third Italian national survey on knowledge, attitudes, and sexual behavior in relation to HIV/AIDS risk from 2002 (Signorelli and Colzani, 2007; Signorelli et al., 2006). Screening characteristics assumed in the model were those of the local organized cervical cancer screening program, active since 1992 and targeting women between 25 and 64 years of age (Ronco et al., 1997). Since the late 1970s and early 1980s, women in the area of Turin spontaneously attended cervical screening facilities, and from 1992, they were invited on a regular basis, every 3 years for Pap smear. The results of a randomized controlled trial showed that the sensitivity of the screening program for CIN2+ detection can be estimated around 75% (Ronco et al., 2006a,b). Based on previous studies (Ronco et al., 1997), we have assumed that currently some 15% of the target population has never attended any screening. Between 1992 and 2006, the time interval under investigation, no major changes in the screening protocol and methods have been introduced; thus, screening performance indicators have been kept unchanged in the model (Table 1). Only about 4% of women reported sexual intercourse by the age of 14 (Signorelli and Colzani, 2007), so we assumed that sexual debut in the population did not occur before the age of 15. The force of infection (per capita incidence of infection) for each sex, sexual activity and age group, and the assortativeness of sexual mixingthat we allowed to vary from fully assortative (parameter's value = 0) to random (parameter's value = 1)were calculated using the method proposed by Garnett and Anderson (1994). The best estimates of the assortativeness of mixing by age and sexual activity group and of the probability of transmission per partnership were identied by comparing the predicted and observed distribution of HPV-16 infections and precancerous lesions and by maximizing their log likelihood (Clayton and Hills, 1993). The probability of infection transmission and the assortativeness both by age and class of sexual activity, considered together, were explored simultaneously. Subsequently, age class and sexual activity assortativeness have been explored separately, keeping the best estimate of probability of infection unchanged. Furthermore, to assess the sensitivity of the estimates to changing together age and class of sexual activity assortativeness, selected combinations of discordant values of assortativeness by age and class of sexual activity have been tested by varying the probability of infection. To calculate the log-likelihood of HPV-16 infection prevalence estimates, binomial distribution has been assumed (i.e., log-likelihood = D log(p) + (N D) log(1 p), where D is the number of women who get infected, N is the total number of women at risk of being infected, and p is the estimated prevalence), whereas for the CIN2+ incidence estimates, Poisson distribution has been assumed (i.e., loglikelihood = D log(l) (py l), where D is the number of women who develop a precancerous lesion, py is the total observation time, and l is the estimated incidence rate), the likelihoods of both estimates have then been added together.

Rates of clearance and progression of infection and precancerous lesions Most HPV-16 infections are transient, and observational studies have shown that a signicant proportion of women with HPV infections do not develop detectable serum antibodies (Bontkes et al., 1999; Carter et al., 2000; de Gruijl et al., 1997; Frazer, 2009; Ho et al., 2004), with failure to seroconvert to HPV-16 being associated with having had only one HPV DNA-positive visit (Carter et al., 2000). Furthermore, it is unclear under which condition reinfection with the same HPV type can occur (Trottier and Franco, 2006). Detection of HPV-16 DNA in consecutive samples has been shown to represent both reinfection with the same viral type (Mayrand et al., 2000) and persistent infection lasting several years (Castle et al., 2005; Herrero et al., 2005). Thus, we have assumed that transient HPV-16 infections (i.e., lasting less than 1 year) generate immunity to subsequent reinfection in a fraction of the infected women; furthermore, to account for the possibility of developing type-specic immunity as a consequence of repeated infections with HPV-16, we have assumed that the probability of developing a complete and lifelong immunity increased as a function of a woman's age (Castle et al., 2005; Woodman et al., 2007). Similar modeling assumptions have been necessary to provide a good t in other HPV modeling exercises (Kim et al., 2007a,b; Van de Velde et al., 2007) and allowed us to generate appropriate age-specic patterns of infection from available behavioral and prevalence data. On the other hand, we have assumed that all persistent HPV-16 infections (i.e., lasting more than 1 year), if and when cleared, develop protective immunity. Finally, age-specic cervical cancer mortality was obtained from the reports published by the local cancer registry rates. The model does not account for the potential effect of viral load on precancerous lesions occurrence. We assumed that duration of infection and precancerous lesions affected their respective rates of progression and clearance and we systematically explored, by comparing the t between model's estimates and empirical data, how different assumptions about (a) the weight of duration of infection affected the rates of progression and clearance of the infection itself and (b) the weight of woman's age and duration of CIN2+ lesions affected the rates of the clearance and progression rates of precancerous lesions. Given the large number of possible combinations between CIN2+ clearance and progression rates as a function of both women's age and duration of CIN2+ lesions, we restricted our exploration to linear and exponential relationships. Over numerous iterations, we identied only three sets of combinations of CIN2+ clearance and progression patterns that were compatible to the data. Thus, we restricted our explorations of best combinations of parameters within these scenarios. The best combinations of parameters as a function of women's age and duration of CIN2+ lesions were assessed by calculating the maximum log likelihood (Clayton and Hills, 1993) comparing the predicted and observed distribution of cervical cancers. To calculate the likelihood of cervical cancer incidence estimates, the incidences observed in the two periods (19982001 and 20002002, respectively) have been averaged, and a Poisson distribution has been assumed to t the averaged cervical cancer incidence. Model tting and validation

Table 1 Assumed characteristics of screening program in Turin. Screening characteristic Fraction of the population never screened Age range covered by the screening program Screening program sensitivity Screening interval Screening method Value 15% 2564 75% 3 years Pap smear

The model's outputs were compared with the following agestratied data: (a) the prevalence of HPV-16 infection estimated for Turin (Ronco et al., 2005; Ronco et al., 2006b), (b) the age-specic incidence of CIN2+ cases, detected by the local organized screening programs between 2001 and 2006 (unpublished data), and (c) the incidence of cervical cancer, reported by the cancer registry of Turin, for the periods of 19982001 (AIRTUM Working Group, 2007) and 20002002 (CPO Piemonte, 2007). The proportions of CIN2+ lesions

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Fig. 2. Comparison between observed data and model's estimates. (A) Observed and estimated prevalence of HPV-16 infection by age in Turin(Ronco et al., 2005). (B) Observed and estimated incidence of CIN2+ (HPV-16+) by age (Turin 20012006, unpublished data from the local screening program). The proportions of CIN2+ lesions were calculated from the estimates for the Southern Europe (Smith et al., 2007).

and cervical cancers that are HPV-16-positive were calculated from estimates for Southern Europe (Smith et al., 2007). We validated the model outputs by assessing the consistency between our estimates of the transition rates and those reported from observational studies and the consistency between our estimates of cervical cancer incidence, assuming absence of screening, and the estimates reported in the literature (Gustafsson et al., 1997; Mandelblatt et al., 2002; Myers et al., 2000; Peto et al., 2004). Results Overall, the model was able to represent the age-specic observed distribution of HPV-16 infection, lesions, and cancers (Figs. 2 and 3). Our maximum likelihood estimate of the probability of transmission per sexual partnership was 40%, and we found that the transmission

of HPV-16 infection occurred within a mildly assortative sexual mixing context: the parameters specifying assortative mixing by age and by classes of sexual activity were 0.4 in both cases (where 0 is fully assortative and 1 is random). This means there is some, but not complete, correlation in ages and behaviors of sexual partners. The rates of HPV-16 infection clearance and progression to precancerous lesions declined exponentially as a function of the time elapsed since infection (the duration of infection). In particular, the rate of progression of HPV-16 infection toward the occurrence of precancerous lesions was 0.072 per woman-year in the rst 6 months of infection, decreasing at 0.018 after more than 6 years of infection; similarly, the rate of clearance of HPV-16 infection decreased from 1.6 to 0.036 per woman-year during the same period. Our ndings are consistent with the reported estimates of the rates of clearance (Giuliano et al., 2002; Koshiol et al., 2006; Moscicki et al., 2004; Munoz et al., 2004; Richardson et al., 2003; Rodriguez et al., 2008; Schlecht et al., 2003; Woodman et al., 2001; Xi et al., 2002) and progression (Ho et al., 1998; Khan et al., 2005; Rodriguez et al., 2008; Wheeler et al., 2006; Winer et al., 2005; Woodman et al., 2001; Ylitalo et al., 2000a,b) of HPV-16 infection. We identied three patterns of interplay between CIN2+ clearance, possibly the most uncertain parameter (Van de Velde et al., 2007), and progression rate of precancerous lesions to cancer, that could t the CIN2+ and cervical cancer incidence data, described as rapid, intermediate, and slow clearance scenario. In all these scenarios, the rate of clearance of precancerous lesion was found to decrease exponentially with women's age. Our exploration of parameters covered the full range of values for the rate of clearance reported in the literature, both in observational studies and in modeling studies (Giuliano et al., 2002; Kim et al., 2007a,b; Koshiol et al., 2006; Moscicki et al., 2004; Munoz et al., 2004; Richardson et al., 2003; Rodriguez et al., 2008; Schlecht et al., 2003; Van de Velde et al., 2007; Woodman et al., 2001; Xi et al., 2002). For the rapid scenario, precancerous clearance rate ranged between 0.7 and 0.6 per womanyear; for the intermediate scenario, precancerous clearance rate ranged between 0.07 and 0.06 per woman-year; and for the slow scenario, the rate ranged between 0.007 and 0.006 per woman-year.

Fig. 3. Calibration of cervical cancer (HPV-16+) incidence estimates (curves) against observed data (bars) by age (Turin, 19982002), according to the CIN2+ clearance rate scenario. For each clearance scenario, the represented curves show a selection of model's outputs. For each estimate, the t to the observed data has been assessed, the estimate showing the best t to the observed data has been selected to assess the rate of progression of CIN2+ lesions to cervical cancer. (A) Rapid CIN2+ clearance scenario. Each curve represents the estimated age-specic incidence of cervical cancer (HPV-16+), assuming a specic effect of women's age on the CIN2+ progression rate. Overall, the stronger the effect of women's age, the greater the incidence of cervical cancer, whereas the different effects of the precancerous lesion duration (ranging from no effect to maximum explored effect) are undistinguishable and not reported. The simulations assuming lower effect of women's age underestimate the cervical cancer incidence for the extreme age classes, whereas those assuming higher women's age effect overestimate the cervical cancer incidence for the central age classes. (B) Intermediate CIN2+ clearance scenario. Each curve represents the estimated age-specic incidence of cervical cancer (HPV-16+), assuming a specic effect of both women's age and duration of the lesion on the CIN2+ progression rate. The simulations assuming lower effect of women's age and duration of the lesion underestimate the cervical cancer incidence, those assuming higher effect of women's age and duration of the lesion overestimate the cervical cancer incidence, while the simulations assuming an intermediate effect of women's age and duration of the lesion estimate adequately the cervical cancer incidence. (C) Slow CIN2+ clearance scenario. Each curve represents the estimated age-specic incidence of cervical cancer (HPV-16+) assuming a specic effect of duration of the lesion on the CIN2+ progression rate. As soon as women's age effect is accounted for, the t between estimates and observed data worsens (data not shown). The simulations assuming lower effect of duration of the lesion underestimate the cervical cancer incidence, those assuming a higher effect of duration of the lesion overestimate the cervical cancer incidence, while those intermediate are adequate.

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Meanwhile, the rate of progression from precancerous lesion to cervical cancer was found to depend on both women's age and the duration of precancerous lesions. The role of women's age and of the duration of precancerous lesions differed according to the precancerous lesions clearance rate scenario. First, for the rapid clearance of the precancerous lesions, changing the CIN2+ progression rate as a function of the duration of the lesion did not inuence the t between estimated and observed incidence of cervical cancer. Changing the effect of woman's age alone did inuence the predicted incidence but was not sufcient to generate a good match with the observed pattern of cervical cancer incidence (Fig. 3A). Second, for the slow clearance of the precancerous lesions, the best t between estimated and observed cancer incidence was obtained when only the duration of precancerous lesions determined the CIN2+ progression rate, while as soon as the woman's age effect was taken into account, the goodness of t signicantly decreased (Fig. 3C). Finally, for the intermediate scenario of CIN2+ lesions clearance, the t with respect to age overall was reasonable, but there are discrepancies in the t between middle and extreme age groups. The best t between the model's estimates and observed data was obtained when both women's age and the duration of precancerous lesions had an effect on the rate of progression; if the rate was constant with respect to either, the t to the observed data signicantly decreased (Fig. 3B). As expected, the estimated annual per capita rates of the lesion's progression to cervical cancer were higher (between 0.005 and 1.23 per woman-year) for the rapid clearance scenario, lower (between 0.0004 and 0.02 per woman-year) for the slow clearance scenario, and intermediate (between 0.001 and 0.45 per woman-year) for the intermediate clearance scenario. The range of values explored, the methods of calibration, and the model's best estimates of the different parameters regulating the natural history of HPV-16 infection and cervical cancer are summarized in Table 2. The modeled effectiveness of screening, measured as cervical cancer lifetime risk reduction, was assessed by comparing the model's estimates obtained including and excluding cervical cancer screening from simulations. For each of the three scenarios, it was found to be implausibly low (12% to 20%) when there is rapid natural clearance of CIN2+. For the intermediate clearance scenario, it was 60% to 75%, and for the slow clearance scenario, it was 70% to 83%.

Fig. 4. Age-specic incidence of cervical cancer, in the absence of screening (model's estimates), according to the CIN2+ clearance rate scenario. For each presented curve, the rate of progression of precancerous lesions to cervical cancer was chosen as the best-tting estimate obtained through the calibration process represented in Fig. 3.

To indirectly validate our model estimates, under the different scenarios of clearance of precancerous lesions, we estimated, using the best tting set of parameters, the expected age-specic incidence rates of cervical cancer (Fig. 4) and the lifetime risk of cervical cancer in the absence of screening. The estimated peak incidence ranged between 27 and 90 cancer cases per 100 000 women, and the age at peak incidence ranged between 55 and 64 years. The decline of the agespecic risk for cervical cancer after the peak incidence was strongly dependent on the scenario of clearance of precancerous lesions, namely a rapid decline in the incidence was estimated in case of rapid or intermediate clearance, whereas a slow decline was estimated in case of slow clearance of precancerous lesions. The lifetime risk of cervical cancer in absence of screening was 3.7% for the scenario of slow clearance of precancerous lesions, 2.4% for the intermediate scenario, and 0.9% for the rapid scenario. Readers can nd further details about the probability of HPV-16 transmission, sexual mixing patterns, rates of progression and clearance of HPV-16 infection, rates of progression and clearance of precancerous lesions, and screening effectiveness in the Supplementary data.

Table 2 Model's estimates: parameter, method of calibration, range of values explored, and best estimate values. Parameter Age-specic sexual assortativeness Behavioural-specic sexual assortativeness HPV-16 infection transmission probability per sexual partnership Rate of HPV-16 infection clearancea Rate of HPV-16 infection progressiona Rate of CIN2+ lesion clearance
b

Method of calibration Log-likelihood Log-likelihood Log-likelihood Visually tted to the observed data Visually tted to the observed data Visually tted to the observed data

Range Explored 01 01 0.0%100% 0.50.0005 2.00.003 2.00.003

Best t estimate 0.4 0.4 40% 0.072 during the rst 6 months of infection to 0.0018 after more than 6 years of infection 1.6 during the rst 6 months of infection to 0.036 after more than 6 years of infection 0.7 for women younger than 25 years of age to 0.6 for women older than 70 years of age (rapid clearance) 0.07 for women younger than 25 years of age to 0. 6 for women older than 70 years of age (intermediate clearance)d 0.007 for women younger than 25 years of age to 0.006 for women older than 70 years of age (slow clearance)e 0.005 for women younger than 25 years of age to 1.2 for women older than 70 years of age 0.001 for women younger than 25 years of age and precancerous lesions younger than 5 years to 0.45 for women older than 70 years of age and precancerous lesions older than 25 years 0.0004 for precancerous lesions younger than 5 years to 0.02 for precancerous lesions older than 25 years

Rate of CIN2+ lesion progressionc Rate of CIN2+ lesion progression

d

Log-likelihood Log-likelihood

0.00063.0 0.000416

Rate of CIN2+ lesion progressione

a

Log-likelihood

0.000324

Annual per-capita rate decreasing with time elapsed since infection occurrence. Annual per-capita rate decreasing with woman's age. Annual per-capita rate increasing with woman's age. d Annual per-capita rate increasing with woman's age and time elapsed since precancerous lesion occurrence. e Annual per-capita rate increasing with time elapsed since precancerous lesion occurrence.
b c

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Discussion In this article, we present the results of a systematic investigation of the effect of the duration of HPV-16 infection and of CIN2+ lesions on their respective progression and clearance rates. The possibility to t our estimates to sets of data covering the whole natural history of infection and cervical cancer in a well-dened geographic area and chronological period was critical to discriminate between the different hypothetical patterns of progression and clearance at each stage of the development of cervical cancer. We estimated a 40% probability of infection transmission per partnership and a mildly assortative sexual behavior both by age and class of sexual activity. Our estimate is consistent with estimates reported in other recent model-based studies (Barnabas et al., 2006; Burchell et al., 2006; Kim et al., 2007a,b). Our ndings show that the clearance and progression rates of the acquired infections decrease with the duration of HPV-16 infection. This may suggest that important events such as infection clearance and progression are more likely to occur early in the natural history of the disease, consistent with data reported from observational studies (Plummer et al., 2007; Schiffman et al., 2007). The progressive escape from the host's immune surveillance may explain biologically the decreasing rate of HPV infection clearance (zur Hausen, 2002), whereas decreasing rates of progression of HPV infection may result from differences in hostpathogen interactions, for example, determined by the interplay between specic HPV-16 variants and human major histocompatibility complex haplotypes (Terry et al., 1997), eventually leading to different rates of progression toward precancerous lesions. Clearance of precancerous lesions was found to depend on age of women but not on age (duration) of the lesions. The nding of inverse women's age dependency on the rate of clearance of precancerous lesions is consistent with the estimates reported from the British Columbia cohort study (van Oortmarssen and Habbema, 1991) and from a recent modeling analysis performed to support cervical cancer prevention policies in the United States (Goldhaber-Fiebert et al., 2007). We further explored the possibility that the rate of clearance of CIN2+ lesions depended also on lesion duration; however, the tested combination of parameters was incompatible with the observed sets of data. In biological terms, this could be explained by an age-related decline of the efciency of the immune control of precancerous lesions (Derhovanessian et al., 2008). At the moment, however, direct evidence supporting this speculation is lacking. The effect of the duration of precancerous lesions on the progression to cervical cancer was sensitive to the assumptions made about the clearance rate of the precancerous lesions, possibly the most uncertain parameter of the natural history of the cervical cancer (Van de Velde et al., 2007). Our ndings contrast extreme scenarios. On one hand, when HPV-16-positive CIN2+ lesions clearance is assumed to be rapid, the progression to cancer depends only on the women's age. Few lesions last long, so to get the increased incidence of cervical cancer with age, the age at which lesions start has a strong inuence on it progressing. On the other, when the clearance is slow, the progression to cancer depends only on the duration of precancerous lesions: the longer a lesion persists, the more likely, per unit of time, it is to progress to cancer. Most lesions persist and cumulatively increase the risk of progression; if risk also increased with age, there would be a continuous high increase as women get older. Assuming an intermediate rate of clearance, women's age and the duration of precancerous lesions act simultaneously to shape the rate of progression to cancer. The effect of the duration of precancerous lesions on their progression rates to cervical cancer could be a result of epigenetic changes (Woodman et al., 2007), of the increased genetic instability associated with the integration of human HPV-16 in cervical keratinocytes, and of the consequent greater probability of acquiring

the secondary genomic abnormalities that may drive malignant progression (Pett et al., 2004). On the other hand, the effect of women's age could be a proxy of the cumulative exposure to tobacco smoking and of hormonal changes possibly contributing to HPVinduced malignant progression (zur Hausen, 2002). Our ndings about the combination between clearance and progression rate of precancerous lesions lead to very different estimates of the effectiveness of screening and of the rates of precancerous lesion progression toward invasive cervical cancer. The scenario of rapid clearance of precancerous lesions seems to be the least plausible for the following reasons: the estimated rates of progression toward invasive cervical cancer, i.e., very rapid at older ages, under such a scenario were not consistent with those that could be estimated from the literature (Galvin et al., 1955; Holowaty et al., 1999; Luthra et al., 1987; McCredie et al., 2008; Melnikow et al., 1998; Murthy et al., 1996; Ostor, 1993). The estimated screening effectiveness ranging between 12% and 20% is low because screening has an insufcient time to pick up lesions that would progress and is not consistent with both general estimates of cervical screening effectiveness (International Agency for Research on Cancer, 2004) and the indications obtained from the local screening performance audit (80% incidence reduction among compliers vs. noncompliers to screening invitation) (Ronco et al., 2005); nally, both the estimated agespecic incidence and the lifetime risk of cervical cancer in absence of screening were too low to support this scenario. Thus, we can conclude that an exclusive role of women's age in shaping the rate of progression to cancer is unlikely. On the other hand, it is not possible to dismiss entirely women's age as a determinant of the progression to cervical cancer in combination with the duration of precancerous lesions because it is not possible to discriminate between the intermediate and slow clearance rate scenarios. For these scenarios, the estimated effectiveness of screening ranged between 60% and 75% and between 70% and 83%, respectively. The rates of progression toward invasive cervical cancer under both scenario were consistent with those that could be estimated from the literature (Galvin et al., 1955; Holowaty et al., 1999; Luthra et al., 1987; McCredie et al., 2008; Melnikow et al., 1998; Murthy et al., 1996; Ostor, 1993), and the estimated age-specic incidence and the lifetime risk of cervical cancer, in absence of screening, could also support both scenarios (Gustafsson et al., 1997; Peto et al., 2004). The estimated age-specic incidence proles, age peaks, and the shape of incidence decline under the two latter scenarios were consistent with those reported by Gustafsson et al. (1997) who summarized the age-specic incidence from 23 different areas of the world. Finally, in terms of lifetime risk of cervical cancer, our ndings were consistent with those reported in the literature. In particular, Peto et al. (2004) adopted an ageperiodcohort model to estimate the cervical cancer lifetime risk for women in England (about 3%), in the absence of screening. Some limitations should be considered when interpreting the results of this study. Given the computational complexity of the model, we could not perform a full multiparameter calibration process. Thus, the tting process was subdivided in two parts: for the infectious part of the model, we focused on the assessment of the probability of transmission per sexual partnership and the model's estimates were calibrated against the observed age-specic HPV-16 prevalence and CIN2+ incidence distributions, whereas for the carcinogenic part of the model, we focused on the assessment of the rate of progression of CIN2+ toward cancer, while the model's estimates were calibrated against the observed age-specic cervical cancer incidence distribution. The inuence of the other rates of transition has been systematically explored over a wide range of parameters values within plausible limits. Any other tested combination of parameters values was found to be incompatible with observed sets of data.

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There is a great deal of uncertainty about the protection offered by the naturally acquired immunity to reinfection; thus, we postulated that early infections can be cleared without developing immunity, while persistent infections are cleared only by developing protective immunity. This assumptions has been made more or less explicitly by several other authors modeling the natural history of carcinogenic HPV and related precancerous lesions (Jit et al., 2008; Kim et al., 2007a,b). Furthermore, multicalibration exercises could not clarify such an issue (Van de Velde et al., 2007). Unfortunately, now, it is impossible to validate such an assumption. However, we can consider the implications of this assumption and of our assumption that protective immunity is more likely at older ages reecting repeated infections. Without such immunity at older ages to get observed agespecic decline in prevalence, sexual contact would have to decrease more rapidly than observed in behavioral data or HPV infections would have to clear more rapidly as age increase. This would lead to high clearance rates with longer infection but concomitant decreases in progression rates from infection to CIN2+. It should have no effect on our ndings on CIN2+ progression and regression. Finally, for the sake of simplicity, we did not explore the possible effect of acquired immunity waning. The changes in sexual behaviors that have occurred since the late 1960s may have inuenced the distribution of HPV-16 and other viral types infection within the population; however, given the lack of data on changes in sexual behaviors over calendar time, we could not assess its importance. For example, the observed increase of CIN2+ incidence among the 34- to 39-year-old women in Fig. 2, not reected in the contemporaries HPV-16 data, was not predicted, possibly because of the resulting changes in sexual behaviors that occurred in the previous years and not incorporated in the model because it was not captured by the available cross-sectional data on sexual behaviors. The model structure does not account for the demographic changes due to immigration: the prevalence of HPV infection among recent immigrants as well as the mixing patterns with the local population is in fact likely to inuence the distribution of infection, but in this case, the data are not available. Finally, although the present model can be used to explore the natural history of infection with any carcinogenic HPV type, we accounted only for the natural history of HPV-16 infection. We preferred to focus exclusively on HPV-16 type because both local data and published reports about the natural history of other carcinogenic HPV types, both HPV-18 and other high-risk types, are less frequent, incomplete, and inconsistent. The consistency of our ndings with several distinct and independent estimates and observations of the natural history of carcinogenic HPV infection and related precancerous lesions supports the validity of the model presented in this article. In conclusion, the presented model is suitable to take into account the duration of infection and lesion sojourn time; this aspect is expected to be relevant to explore and project the impact of combining different strategies of cervical cancer screening and vaccination against selected HPV types.

Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.epidem.2010.02.002. References
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Acknowledgments The authors would like to thank Elio Riboli, Silvia Franceschi, Carlo Signorelli, and Ruanne Barnabas for thoughtful comments and to acknowledge the role of Fulvio Lazzarato as programming advisor. All authors have agreed to all the content in the manuscript, including the data as presented. I.B. was supported by the Compagnia San Paolo-FIRMS (Center for Experimental Research and Medical Studies). G.G. has acted as consultant for and/or received grants for other studies from Sano Pasteur MSD, Merck, and GSK. None of these companies had any role whatsoever in the current study.

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