Time trends of human papillomavirus type distribution in Italian women with cervical intraepithelial neoplasia (CIN)

Gynecologic Oncology 115 (2009) 262–266

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Gynecologic Oncology 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 / y g y n o

Time trends of human papillomavirus type distribution in Italian women with cervical intraepithelial neoplasia (CIN) Barbara Dal Bello a, Arsenio Spinillo b, Paola Alberizzi a, Stefania Cesari a, Barbara Gardella b, Enrico Maria Silini c,⁎ a b c

Department of Pathology, IRCCS-Fondazione Policlinico San Matteo and University of Pavia, Pavia, Italy Department of Obstetrics and Gynaecology, IRCCS-Fondazione Policlinico San Matteo and University of Pavia, Pavia, Italy Department of Pathology, Azienda Ospedaliero-Universitaria di Parma, Parma, Italy

a r t i c l e

i n f o

Article history: Received 14 July 2009 Available online 26 August 2009 Keywords: Cervix SIL CIN HPV Genotypes Molecular epidemiology

a b s t r a c t Objective. It is assumed that the circulation of HPV types in a population is stable over time although there are limited historical data to support this view. The existence of possible cohort effects in the circulation of HPV types has major implications for vaccination strategies and risk assessment in HPVinfected women. We analysed archival biopsy samples of cervical intraepithelial neoplasia (CIN) to study the distribution of HPV types in Northern Italy over the years 1985–2007. Methods. DNA from formalin-fixed paraffin-embedded cervical biopsies from the years 1985–87 (67 samples) and 1995–97 (92 samples) was HPV-typed by the SPF-10 Lipa assay. Cases were compared with 159 control biopsies from the years 2005–07 matched by patient age and CIN grade. Quantitative PCR was used to compare titres of HPV sequences in DNA extracted from biopsies of the three periods. Type-specific PCR was used to confirm HPV51 and 52 typing by SPF-10 Lipa. Results. HPV51, 52, 53, 56, 58, and 66 were markedly under-represented or undetectable in samples from past periods whereas they represented 5.7–30.8% of present infections. Frequency of multiple HPV infections and high-risk infections (p = 0.0001) also increased in recent years. The main changes occurred over the last decade. Infections by HPV16, 18, were three times more frequent 20 years ago than today (p = 0.012). Loss of amplifiable HPV sequences over prolonged storage was not observed. Type-specific PCR confirmed all HPV51 and 52 infections. Conclusions. Secular trends in the distribution of HPV types among women with CIN may occur in specific populations. © 2009 Elsevier Inc. All rights reserved.

Introduction Over 100 human papillomavirus (HPV) types are involved in the etiology of squamous epithelial lesions at different sites; at least 30 of them are found in the cervix [1]. The type and the number of HPVs associated with cervical squamous cell carcinoma and its precursor lesions are variably distributed depending on the characteristics of the population under study, including geographical area, severity of lesions and age strata [2,3]. The reasons to explain geographical differences in HPV type distribution are substantially unknown and specific regional patterns are still poorly defined [4]. Shifts in type distribution according to the severity of cervical lesions are currently explained by the variable ability of the different HPV types to induce persistence and neoplastic changes in the epithelium of the transformation zone [5]. Variations ⁎ Corresponding author. Unità Operativa di Anatomia Patologica, Azienda Ospedaliero-Universitaria, Viale Gramsci 14, 43126 Parma, Italy. Fax: +39 521 292710. E-mail address: [email protected] (E.M. Silini). 0090-8258/$ – see front matter © 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.ygyno.2009.07.029

across age strata are mainly related to acquired immunity and the physiological cervical changes occurring over a woman's lifetime and their impact on the tissue tropism and the persistence of the different HPV types [6,7]. Conversely, it is generally held that HPV type distribution in a population is stable over time although there is a substantial lack of historical data to support this view. There are relevant implications for the estimates of viral oncogenicity in the assumption that HPV types vary according to lesion severity and age because of their biological features rather than as a consequence of time trends in virus circulation. Indeed, the notion of a differential carcinogenic effect across types is at the base of the selective HPV vaccination [8] and of the clinical application of genotyping in the prediction of cervical intraepithelial neoplasia (CIN) progression [9]. We used, SPF-10 Lipa [10], a broad-spectrum, short-fragment PCR assay based on the amplification of a 65-bp fragment of the L1 region of HPV genome, to study HPV genotype distribution in our area over the last 20 years using archival pathological material from punch or cone cervical biopsies. To this aim, Italian women

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Table 1 Main clinical features of women with cervical intraepithelial neoplasia (CIN) in the three periods under study, 1985–87 (1), 1995–97 (2) and 2005–07 (3). Variable

Period (N. of subjects)

Age (SD)a Histology

CIN1 CIN ≥ 2 1 2 3 ≥4 Low-risk Single high-risk Multiple high-risk Multiple low/high-risk

N. of HPV types

Class of oncogenic risk

a b c

p

1 (67)

2 (92)

3 (159)

39.34 (9.26) 18 (26.9%) 49 (73.1%) 28 (41.8%) 28 (41.8%) 9 (13.4%) 2 (2.9%) 6 (9.0%) 24 (35.8%) 29 (43.3%) 8 (11.9%)

35.39 (9.37) 25 (27.2%) 67 (72.8%) 36 (39.1%) 37 (40.2%) 16 (17.4%) 3 (3.3%) 6 (6.5%) 30 (32.6%) 32 (34.8%) 24 (26.1%)

37.04 (9.82) 43 (27.0%) 116 (73.0%) 18 (11.3%) 34 (21.4%) 71 (44.7%) 36 (22.6%) 2 (1.2%) 17 (10.7%) 82 (51.6%) 58 (36.5%)

0.038b 0.868c b 0.0001c

b 0.0001c

Standard deviation. ANOVA. Pearson chi-square.

with histological diagnoses of CIN from two past periods, 1985–87 and 1995–97, were pair-matched with control patients from the years 2005–07. Methods Patients All histological diagnoses of CIN from two periods, 1985–87 (period 1) and 1995–97 (period 2), were retrieved from the archives of the Department of Pathology. Original slides were reviewed to

confirm the diagnosis and to select formalin-fixed paraffin-embedded (FFPE) tissue blocks for DNA extraction. Sixty-seven consecutive cases with a primary diagnosis of CIN from period 1 and 92 from period 2 were considered. For 33 cases from period 2, frozen DNAs from cervical washings collected at the time of biopsy were available. These had been stored during a previous investigation and were used to confirm HPV type attribution in archival samples. Cases from periods 1 and 2 were pair-matched according to age (±2 years) and CIN grade (CIN1 vs. CIN ≥ 2) with 159 FFPE cervical biopsies of HPV-positive women diagnosed in the years 2005–07 (period 3). In all 159 women from period 3, HPV typing was also performed on exfoliated cells

Table 2 Distribution of HPV types and oncogenic categories in the three periods under study, 1985–87 (1), 1995–97 (2) and 2005–07 (3). HPV type

Period 1

HPV 6 HPV 11 HPV 16 HPV 18 HPV 31 HPV 45 HPV 51 HPV 52 HPV 53 HPV 56 HPV 58 HPV 42 HPV 33 HPV 35 HPV 39 HPV 40 HPV 59 HPV 43 HPV 44 HPV 66 HPV 68 HPV 70 HPV 6–11 HPV 16–18 HPV6–11–16–18 Low-risk HPV High-risk HPV

7 4 48 13 24 5 0 0 0 0 0 0 5 2 2 5 2 0 1 0 0 0 9 52 57 14 61

(10.4) (6.0) (71.6) (19.4) (35.8) (7.5) (0) (0) (0) (0) (0) (0) (7.5) (3.0) (3.0) (7.5) (3.0) (0) (1.5) (0) (0) (0) (13.4) (77.6) (85.1) (20.9) (91.0)

Period 2 vs 1

Period 3 vs 1

2

3

Pa

OR (95% CI)b

P

OR (95% CI)b

P

10 (10.9) 7 (7.6) 57 (62.0) 15 (16.3) 30 (32.6) 9 (9.8) 0 (0) 0 (0) 0 (0) 1 (1.1) 1 (1.1) 3 (3.3) 5 (5.4) 1 (1.1) 6 (6.5) 14 (15.2) 1 (1.1) 1 (1.1) 3 (3.3) 2 (2.2) 2 (2.2) 1 (1.1) 16 (17.4) 64 (69.6) 70 (76.1) 30 (32.6) 86 (93.5)

33 (30.8) 16 (10.1) 94 (59.1) 35 (22.0) 70 (44.0) 7 (4.4) 43 (27.0) 49 (30.8) 9 (5.7) 19 (11.9) 12 (7.5) 3 (1.9) 11 (6.9) 7 (4.4) 7 (4.4) 13 (8.2) 2 (1.2) 1 (0.6) 5 (3.1) 6 (3.8) 5 (3.1) 3 (1.9) 46 (28.9) 100 (62.9) 122 (76.2) 60 (37.7) 157 (98.7)

0.048c 0.604 0.204d 0.548 0.169 0.242 b 0.001 b 0.001 0.010 b 0.001 0.007 0.328 0.857 0.348 0.563 0.148 0.575 0.693 0.758 0.247 0.339 0.501 0.015e 0.089f 0.31 0.048g 0.018h

1.14 0.76 0.35 0.57 1.12 7.75 NA NA NA NA NA NA 0.72 0.25 2.51 2.95 NA NA 5.87 NA NA NA 1.30 0.37 0.31 1.70 NA

(0.29–4.52) (0.15–3.93) (0.12–0.99) (0.19–1.77) (0.48–2.61) (0.77–78.4)

0.852 0.741 0.048 0.333 0.801 0.083

(0.83–7.48) (0.33–4.58) (0.15–0.87) (0.42–2.15) (0.80–2.94) (0.30–3.77)

0.102 0.766 0.024 0.910 0.198 0.916

(0.11–4.55) (0.01–5.39) (0.25–24.9) (0.60–14.5)

0.724 0.376 0.433 0.183

(0.23–3.59) (0.17–16.5) (0.24–8.84) (0.36–3.96)

0.897 0.656 0.674 0.762

(0.19–178)

0.310

(0.17–28.4)

0.547

(0.41–4.14) (0.13–1.05) (0.11–0.87) (0.65–4.43)

0.659 0.061 0.026 0.279

2.50 1.22 0.37 0.95 1.53 1.07 NA NA NA NA NA NA 0.91 1.68 1.47 1.20 NA NA 2.20 NA NA NA 2.49 0.33 0.44 2.11 NA

(0.96–6.42) (0.14–0.78) (0.20–0.97) (0.99–4.49)

0.059 0.012 0.041 0.053

NA, not assessable. a Pearson chi-square. b As obtained by conditional logistic regression analysis including terms for age and period. c p for trend 0.021. d p for trend 0.05. e p for trend 0.009. f p for trend 0.018. g p for trend 0.04. h p for trend 0.034.

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obtained by scraping at the time of biopsy. Non-Italian descent and HIV infection were criteria of exclusion. The main clinical features of the cases are detailed in Table 1. All histological diagnoses were rendered by the same experienced gyneco-pathologists (EMS and BDB), in most cases by consensus reading, and were reported according to the 2004 WHO system [11]. The study was approved by the local Institutional Review Board. HPV-DNA detection and typing For DNA isolation from FFPE samples, 3–5 10 μm sections were incubated in 200 μl of a lysis solution (1 mg/ml Proteinase K in 50 mM Tris, pH 8.0, 1 mM EDTA, and 0.45% Tween-20 and 0.45% IGEPAL CA630) for 16/24 h at 56 °C. Proteinase K was heat inactivated, the lysates were centrifuged to eliminate wax, extracted with phenol– chloroform and resuspended in 100 μl. DNA extraction by cervical scraping was performed as previously described [12]. Appropriate positive and negative controls were introduced in each set of reactions and blank reagents were used throughout all steps of the procedure. HPV type-specific sequences were detected by the line probe, INNO-LiPA HPV genotyping assay (Innogenetics N.V., Ghent, Belgium) [10] according to the manufacturer's instructions. The assay allows the simultaneous and separate detection of 15 high-risk (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68, and 70), and 10 low-risk HPV types (6, 11, 34, 40, 42–44, 53, 54, and 74). Two sets of PCR primers were designed for the specific amplification of sequences from the L1 ORF of HPV 51 (forw 5′-ACTTGTAGGTGTTGGGGAAG-3′; rev 5′-AACTTTCTATAGGGTCACGG-3′, 100 bp) and HPV52 (forw 5′-AGGAATACCTTTCTGTATGGC-3′; rev 5′CTCTAAAATAGTGGCATCCATC-3′, 80 bp). Amplification was performed for 35 cycles at an annealing temperature of 60 °C. The specificity of the amplification procedure was assessed by gel sizing, restriction enzyme analysis and direct sequencing of PCR products.

and 159 from period 3 (2005–07) for a total of 737 individual HPV infections (Table 1). In 15 women from periods 1 and 2, typing was performed on both punch and corresponding cone biopsy and was confirmed in 36 of 38 (94.7%) individual HPV types. In 33 women from period 2, frozen DNA samples from cervical washings obtained at the time of biopsy were available and allowed to confirm typing in 73 of 74 (98,6%) individual infections. The agreement for the number of HPV types between cervical biopsy and scraping in the 159 women of period 3 was 76.7%. Out of a total of 477 individual HPV infections, 37 (7.8%) were detected only in biopsy and 10 (2.1%) only in scraping. These results are consistent with our previously reported data [12]. Overall, the number of infections by multiple HPV types (p b 0.0001) and high-risk types (p for trend 0.034) was higher in period 3 compared to periods 1 and 2 (p b 0.0001) (Table 1). Infections by HPV51, 52, and 53 were not observed in CIN specimens from periods 1 and 2 whereas they were present in period 3 at a frequency of 5.7–30.8%. A significantly lower prevalence of infections by HPV6, 56, 58 and 66 was also observed (Table 2). The probability of individual infections for past compared to present time periods was calculated by a case-control analysis matching for age and CIN grade. Since after matching the mean age of the patients in the three periods was slightly unbalanced, age was inserted as confounder in conditional logistic regression (Table 2). The odds ratio for HPV16 infection was approximately one third in periods 2 and 3 compared to period 1 (p 0.48 and 0.024, respectively; p for trend 0.06). The frequency of HPV16 started to decline in period 2 although the number of infecting types was comparable to previous years and there was an overall increase of high-risk infections (Table 1). Infections by HPV16– 18 were three times less frequent in periods 2 and 3 compared to period 1 (p for trend 0.018), whereas a corresponding increase of HPV6–11 infections was observed (p for trend 0.009). Overall, infections by HPV6–11–16–18 were three times less frequent in periods 2 and 3 compared to period 1 (p 0.026 and 0.041, respectively) (Table 2).

Quantification of HPV-DNA One hundred and seventy-three FFPE biopsies from the three periods were considered for quantitation of HPV-DNA sequences. Amplifiable HPV-DNA sequences were assessed by real-time quantitative PCR on a Mx300 thermal cycler (Stratagene M3000P, M Medical/MGW Biotech, Milan, Italy). DNA extracted from FFPE histological sections containing a comparable amount of cervical epithelium as assessed by microscopy, were amplified with SPF-10 primers using the Platinum SYBER Green qPCR super MIX kit (Invitrogen SRL, Milan, Italy). A dilution series of Siha and HeLa DNA with a known number of copies were used as calibration standard for each experiment. Each sample was analysed in triplicate. HPV-DNA titres were expressed as number of copies. Statistical analysis Statistical analysis was carried out with one-way ANOVA and chisquare test to compare continous and categorical variables, respectively. Spearman rank correlation coefficient was used to test for linear trend with increasing exposure. Conditional logistic regression on matched sets was used to compute exact odds ratios associated with the different types of HPV infection in period 2 and 3 compared to 1. Statistical analysis was performed by the software package STATA 10.0. Results Formalin-fixed paraffin-embedded (FFPE) cervical biopsies from 318 women with a primary diagnosis of CIN were examined by SPF-10 LIPA, 67 were from period 1 (1985–87), 92 from period 2 (1995–97)

Fig. 1. Quantification of HPV sequences by real-time PCR with SPF-10 primers in 173 formalin-fixed paraffin-embedded cervical biopsy specimens from three time periods, 1985–87 (1), 1995–97 (2) and 2005–07 (3).

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Broad-spectrum typing assay such as SPF-10 Lipa do not have the same specificity and sensitivity for each viral sequence [13], this may affect the representation of types because of competition for PCR primers. To rule out this possibility, two type-specific primer sets were designed to amplify short sequences in the L1 ORF of HPV51 and HPV52. These two HPV types were absent in periods 1 and 2 whereas they represented 27.0% and 30.8% of all HPV types in period 3, respectively. Eleven HPV51-positive samples (3 single and 8 multiple infections) and 16 HPV52-positive samples (4 single and 12 multiple infections) were amplified with the two type-specific primer sets. In all cases, the presence of specific sequences was confirmed whereas these were not detected in any of 45 HPV51-negative and 58 HPV52negative FFPE controls. Degradation of DNA may occur with long-term storage of FFPE samples and affect the representation of HPV types present at low titres [14]. To exclude this possible bias, similar quantities of DNA extracted from 49 FFPE samples from period 1, 50 from period 2 and 74 from period 3 were quantified by real-time PCR using SPF-10 primers. No significant differences in mean titres were observed between periods (log10 5.22 period 1, 5.223 period 2, 5.414 period 3; p 0.0865) (Fig. 1). Mean titres of amplifiable HPV sequences were similar according to CIN severity (p 0.9759) and number of HPV types (p 0.4419). Discussion A change in the distribution of HPV types among women with a histological diagnosis of CIN was observed in our area over the period 1985–2007. HPV genotypes that were rare or undetectable 10 to 20 years ago, namely HPV51, 52, 53, 56, and 58, can now be observed in over one third of CIN cases. The main changes seem to have occurred over the last 10 years, whereas the distribution of HPV infections was similar in the periods 1985–87 and 1995–97. An increase in the frequency of multiple type and high-risk type infections was observed over time. The four HPV types targeted by current multivalent vaccines, HPV6, 11, 16 and 18, are two to three times less frequent today than in the past two decades. Archival pathological material is a valuable source to assess microbial diversity across geographical areas and time periods and it has been instrumental in the definition of the etiologic role of HPV in cervical cancer [15]. However, few studies have used this approach to investigate secular changes in HPV type distribution. Previous studies did not provide consistent results as they have examined small and selected cohorts from isolated areas [16,17]. Furthermore, these studies used non-validated molecular tests and did not control for confounding. Besides allowing the retrieval of past pathologic material, the study of FFPE tissue allows to target the diseased epithelium as compared to cytological sampling that may reflect unselected cervical areas or vaginal infections [18]. The SPF-10 Lipa assay used for the analysis is a highly validated test with the potential to simultaneously identify 54 independent HPV types [10]. It has proven clinical significance and has been widely used in clinical and epidemiological studies [19–21]. SPF-10 Lipa employs the smallest amplicon of any HPV-DNA typing system available [22], for this reason it is thought to be more sensitive to amplify HPV-DNA from FFPE samples as the PCR efficiency is inversely correlated to the length of the amplicon [23]. Comparative performances with other tests have confirmed reproducible accuracy and high sensitivity in typing of single and multiple HPV infections [24–26]. The use of SPF-10 Lipa in HPV typing of FFPE material had been previously validated [27]. We previously compared HPV typing in 165 paired samples of cervical scraping and biopsy and calculated a 95.4% agreement. Biopsy performed better likely due to a more effective sampling of the lesion and the endocervix [12]. These results are confirmed by the present study. SPF-10 amplifiable HPV sequences from FFPE samples of different periods were also quantified by real-time PCR to exclude

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that long-term storage or different fixation or embedding procedures might have caused a drop in viral titres below the sensitivity threshold of the assay [14]. Finally, HPV typing in FFPE biopsy samples from the 1985–87 and 1996–97 years was confirmed with high reproducibility by repeat testing on cone biopsy specimens and frozen DNA from cervical washings. Broad-spectrum PCR may be affected by competition among viral sequences at the primer level causing underestimation of multiple infections especially when minority types are present at low relative concentrations [13]. Previous studies have shown that SPF-10 Lipa may have lower analytic sensitivity compared to type-specific PCR, although significant biases in amplification or detection of the specific HPV types differentially represented in the current study have never been reported [24–26,28]. SPF-10 Lipa typing of the two genotypes with the largest differences in prevalence over time, HPV51 and HPV52, was faithfully reproduced by type-specific PCR in 130 samples examined; this excludes biases in sensitivity and specificity of the typing assay. A recent study from the New Mexico used an experimental approach similar to that of the present study to show that the prevalence of HPV16 in cervical squamous cell carcinoma (SCC) has declined over the period 1980–1999 [29], an observation that is entirely in agreement with our findings. SCC is a stronger outcome than CIN when seeking evidence for secular trends in the circulation of oncogenic HPVs. However, population screening for cervical precancerous lesions might result in censoring of some viral types in SCC; this is less likely if first diagnoses of CIN are considered as in the present study. Differences between groups were further controlled by pairmatching for the three main variables known to affect HPV distribution, country of origin, age and CIN severity. Nonetheless, it must be acknowledged that the retrospective design of the study has limitations that may have affected HPV type representation between groups. First, the prevalence, the clinical awareness and the screening practices of precancerous lesions were different in the past. Secondly, the distribution of HPV types in the referral population over the periods of study is unknown. Finally, residual confounding due to unaccounted behavioural and socio-economical factors cannot be excluded. Assuming that our observations reflect a bona fide change in HPV type circulation, this can be explained by two non-mutually exclusive hypotheses: i) ‘new’ viral types have been introduced from other geographic areas; ii) endemic types have undergone a redistribution due to changes in the patterns of exposure. Prevalence and type distribution of HPV infections vary greatly across geographical areas [3]. Both meta-analyses [30,31] and international multicentric studies [32,33] indicate that HPV16 and HPV18 are most commonly involved in CIN/SCC worldwide, but the next most frequent types, HPV31, 33, 45, 52, 58, 35, show significant regional variation. Thus, HPV35 is more frequent in Africa, HPV52 and HPV-58 in Asia, and HPV31 is common in Europe. Cross-sectional studies indicate that these regional variations are consistent across age strata although odds ratios are higher for younger classes [31]. As a consequence of immigration or higher population mobility in recent years, a new pool of viruses might have been introduced in our area [34,35]. However, it should also be acknowledged that most studies of HPV type prevalence in specific populations or in pooled data did not report a relevant cohort effect in type distribution once accounted for disease severity [4]. It is possible that the high level of confounding related to age including sexual activity, physiological changes at transformation zone, acquired immunity, changing rates of persistence, variable penetrance of screening might require very large samples to detect differences in virus distribution across age strata. These arguments do not exclude that a shift in the distribution of an endemic pool of HPV types may have occurred as a consequence of changes of social and behavioural variables. A relevant precedent can be found in the variation of hepatitis C virus genotype distribution associated with the spread of intravenous drug abuse [36]. Indeed, the

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higher number of multiple infections and high-risk HPVs observed in recent years might indicate increased or changing patterns of exposure. Some epidemiological observations confirm an increase in seroprevalence and high-risk HPV infections in recent years [37–39]. Another variable potentially able to influence the infecting HPV types is the immune status. Some genotypes might be more sensitive to reactivation or re-infection following HIV infection or immune defects secondary to infections, malnutrition, age of acquisition, or promiscuity. Notably, high-risk genotypes other than HPV16 are more often found in HIV-infected women and are frequently associated with abnormal cytological findings [40]. In the US, the most prevalent genotypes among HIV-positive women are HPV56 and 53 in lowgrade and HPV52 and 58 in high-grade lesions at variance with HIVnegative women [41]. In conclusion, the present results suggest that secular trends in the distribution of HPV types may occur possibly reflecting the introduction of new HPV types and/or changing patterns of exposure. This has major implications for prevention policies and risk assessment in HPV-infected patients and warrants further investigation. Most importantly, our results should be confirmed in independent cohorts and considering general population samples of larger size and over longer time intervals. Conflict of interest statement No conflict of interest is declared.

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This research was partially supported by grant RC805036 from the Italian Health Ministry to the IRCCS-Fondazione Policlinico San Matteo, Pavia.

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