International Journal of Gynecology and Obstetrics 125 (2014) 107–110

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CLINICAL ARTICLE

Characterization of human papillomavirus genotypes and HPV-16 physical status in cervical neoplasias of women from northern Portugal Joana Ribeiro a,b,c,d, Dulce Teixeira a, Joana Marinho-Dias a,b, Paula Monteiro e, Joana Loureiro e, Inês Baldaque a, Rui Medeiros a,b,d, Hugo Sousa a,b,⁎ a

Virology Service, Portuguese Institute of Oncology, Porto, Portugal Molecular Oncology Group, Portuguese Institute of Oncology, Porto, Portugal Faculty of Medicine, University of Porto, Porto, Portugal d Research Department, Portuguese League Against Cancer (Liga Portuguesa Contra o Cancro – Núcleo Regional do Norte), Porto, Portugal e Department of Pathology, Portuguese Institute of Oncology, Porto, Portugal b c

a r t i c l e

i n f o

Article history: Received 12 July 2013 Received in revised form 17 October 2013 Accepted 10 January 2014 Keywords: Cervical cancer HPV-16 Human papillomavirus genotypes Integration

a b s t r a c t Objective: To determine human papillomavirus (HPV) genotypes and the physical status of HPV-16 DNA among women from northern Portugal with cervical lesions. Methods: The present retrospective study included samples of cervical exfoliated cells from 88 women (median age 42.0 ± 13.1 years) who attended the Gynecology Service at the Portuguese Institute of Oncology in Porto during 2010. After DNA extraction, HPV genotyping was performed by polymerase chain reaction (PCR) followed by restriction fragment length polymorphism analysis using the MY09/MY11 primers. The physical status of HPV-16 was determined by real-time PCR. Results: Overall, 69.3% of the samples tested positive. The prevalence of HPV infection was 38.5% in normal samples, 57.7% in cervicitis samples, and 87.2% in all cervical lesions including invasive cancers. Sixteen genotypes were detected, the most prevalent ones being HPV-16 (42.9%), HPV-31 (12.2%), and HPV-58 (10.2%); HPV-18 was rare. The overall prevalence of HPV-16 integration was 31.6%. The physical status of HPV-16 did not differ significantly by histology. Conclusion: The most frequent genotypes were HPV-16, -31, and -58. Integration of HPV-16 DNA seemed to be an early event in cervical carcinogenesis. Further studies are required to clarify the value of viral integration as a prognostic marker. © 2014 International Federation of Gynecology and Obstetrics. Published by Elsevier Ireland Ltd. All rights reserved.

1. Introduction Since the early 1990s, persistent infection by high-risk human papillomavirus (HPV) has been recognized as a necessary but not sufficient condition for the development of cervical cancer [1,2]. Human papillomavirus infection is highly prevalent among sexually active women. Most infections regress spontaneously, but a small percentage persists leading to development of cervical lesions that can progress to invasive carcinoma [3]. Pap smear testing is the most commonly used screening procedure throughout the world for the diagnosis of cervical lesions. Nevertheless, some countries have already implemented the detection of HPV DNA [4,5]. Genotyping of HPV allows the identification of women with persistent infection and consequently with an increased risk for malignant progression. Since the development of genotyping techniques, many studies [6–8] have described the distribution of HPV genotypes in specific populations, which is useful to improve the clinical approach and to guide vaccination programs. In addition, assessment of the physical ⁎ Corresponding author at: Serviço de Virologia, Laboratórios Piso 4, Instituto Português de Oncologia do Porto FG, EPE, Rua Dr. António Bernardino Almeida, 4200–072 Porto, Portugal. Tel.: +351 22 508 4000x5410; fax: +351 22 508 4001. E-mail address: [email protected] (H. Sousa).

status of HPV DNA (episomal versus integrated) has been suggested as a tool to identify women at increased risk of cervical cancer [9,10] because viral integration into the host genome is considered to be a hallmark of HPV-associated carcinogenesis. However, the clinical significance of detecting the physical status of HPV DNA remains unclear. To date, few studies [11–15] have characterized the distribution of HPV genotypes in Portuguese populations. The present study aimed to describe the HPV genotype profile among women from the Norte region of Portugal and to determine the physical status of HPV DNA among women with HPV-16 infection. 2. Materials and methods A retrospective hospital-based study was performed with samples of cervical exfoliated cells from 88 women (median age 42.0 ± 13.1 years) from northern Portugal. The women were attending routine clinical visits at the Gynecology Service of the Portuguese Institute of Oncology of Porto, Portugal, between January 1 and December 31, 2010. All samples were submitted to cytologic examination and the results were confirmed by biopsy. Experienced pathologists classified the samples histologically as normal (n = 13; median age 49.0 ± 11.0 years), cervicitis (n = 26; median age 44.0 ± 12.8 years), cervical intraepithelial

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neoplasia (CIN) grade 1 (n = 20; median age 37.5 ± 9.43 years), CIN 2 (n = 7; median age 38.0 ± 9.25 years), CIN 3 (n = 12; median age 45.5 ± 10.8 years), carcinoma in situ (CIS; n = 4; median age 48.0 ± 19.5 years), and invasive cervical carcinoma (ICC; n = 4; median age 75.0 ± 20.8 years). All samples had been collected for the purpose of routine HPV detection and characterization at the Virology Service, Portuguese Institute of Oncology in Porto. Therefore, ethics approval was not required. Cervical exfoliated cells were collected in ThinPrep (Hologic, Bedford, MA, USA) tubes and stored at room temperature prior to processing. An aliquot of 1 mL was used for nucleic acid extraction using the High Pure Viral Nucleic Acid Kit (Roche Applied Science, Mannheim, Germany) according to the manufacturer’s instructions. The quality of DNA/RNA was assessed by measuring the absorbance at 260/280 nm, and the presence of amplifiable genomic DNA was tested by amplification of a region from the β-globin gene as previously described [16]. Human papillomavirus DNA was detected as previously described [16] through polymerase chain reaction (PCR) using 2 sets of consensus primers that amplify highly conserved regions of the HPV L1 gene and are potentially capable of detecting a large number of mucosal HPV types in a single PCR: primers GP5+ and GP6+, which amplify a region of 150 bp; and the degenerate primers MY09 and MY11, which amplify a region of 449–458 bp depending on the HPV type. The detection of HPV DNA was performed in duplicate to evaluate the efficiency of the amplification; the results were confirmed by comparison of the 2 PCR products and the samples were retested if different results were found. Samples testing positive for HPV DNA were genotyped using the protocol and algorithm described by Nobre et al. [17], and the HPV types were grouped as high-risk types (HPV-16, -18, -31, -33, -35, -39, -45, -51, -52, -56, -58, and -59), probable high-risk types (HPV-26, -53, -66, -68, -73, and -82), low-risk types (HPV-6, -11, -13, -40, -42, -43, -44, -54, -55, -61, -70, -72, -81, and -89), and types of undetermined risk (HPV-30, -32, -34, -64, -62, -67, -69, -71, -74, -83, -84, -85, -86, -87, -90, -91, -97, -102, and -106) [11]. The physical status of HPV-16 DNA was analyzed using a multiplex real-time PCR protocol that allows simultaneous amplification of the E2 and E6 regions, adapted from a protocol described by Cañadas et al. [18]. The reaction was performed in a volume of 25 μL, containing 200 ng of DNA, 1 × TaqMan Universal Master Mix II (Applied Biosystems, Foster City, CA, USA), 30 pmol of E2 forward and reverse primers, 6 pmol of an E2 probe, 3 pmol of E6 primers, and 1 pmol of an E6 probe. The amplification conditions included 1 step at 50 ºC for 2 minutes, followed by 45 cycles of 95 ºC for 1 minute, 55 ºC for 1 minute, and 72 ºC for 1 minute. The physical status (episomal, integrated, or both) of HPV-16 DNA was determined based on the principle that the E2 gene is partially or totally disrupted when integration occurs, whereas the E6 gene remains intact [18]. We calculated the ratio of the cycle thresholds obtained by E2 and E6 amplification; an E2/E6 ratio of 1 or more indicated the presence of episomal DNA only; an E2/E6 ratio of less than 1 indicated the presence of both episomal and integrated DNA; and an undetectable level of E2 amplification indicated the presence of integrated DNA only. To ensure the quality of the PCR data, all PCR reactions included a negative control (double distilled water instead of template DNA) and a positive control (a sample from the external quality control panel of the Virology Service, which contains different HPV genotypes). All results were confirmed independently by 2 researchers. The statistical analysis was performed with SPSS version 20.0 (IBM, Armonk, NY, USA). Frequencies were compared with the t test or the χ2 test as appropriate. P b 0.05 was considered statistically significant. Odds ratios (OR) and their 95% confidence intervals (CI) were calculated to measure the association between categorical variables. 3. Results The overall frequency of HPV infection was 69.3% (61/88); 2.3% (2/88) of the results were inconclusive.

Fig. 1. Age-stratified human papillomavirus distribution (n = 86).

Human papillomavirus infection was more common (87.5%) among women below the age of 35 years than among women aged 35–45 years (69.0%), those aged 45–55 years (61.9%), and those who were older than 55 years (58.3%) (Fig. 1). Despite the observed differences, there was no statistically significant association between HPV infection and age below 45 years (P = 0.089; OR 2.26 [95% CI, 0.88–5.82]) or age below 35 years (P = 0.054; OR 2.96 [95% CI 0.91–9.74]). Stratification of the HPV infection data by cervical histology revealed that HPV infection was present in 5/13 (38.5%) samples classified as normal, 15/26 (57.7%) samples classified as cervicitis, 15/20 (75.0%) samples classified as CIN 1, 6/7 (85.7%) samples classified as CIN 2, 16/ 16 (100.0%) samples classified as CIN 3/CIS, and 4/4 (100.0%) samples classified as ICC (Table 1). The 2 samples with inconclusive results were classified as cervicitis and ICC, respectively. Given that the rate of HPV infection did not differ significantly (P = 0.257) between the normal and the cervicitis samples, these were grouped together to form the reference group in the odds ratio analysis. The analysis revealed a strong association between HPV infection and the development of cervical intraepithelial lesions including ICC (OR 6.49 [95% CI, 2.24–18.8]; P b 0.001) (Table 1). In an analysis adjusted for age, the results did not deviate significantly from the non-adjusted results (data not shown). Genotyping of HPV was possible for 49/61 (80.3%) of the HPVpositive samples; for the remaining samples, the PCR amplification yield was too low (n = 8) or the restriction fragment length polymorphism pattern was ambiguous (n = 4). In the total population, 16 different genotypes were identified, including 9 high-risk types, 4 probable high-risk types, and 3 low-risk types (Table 2). High-risk HPV genotypes were more common (81.6%) than probable high-risk genotypes (8.2%) or low-risk genotypes (6.1%) (Table 3). The most common genotype in single infections was HPV-16 (40.8%), followed by HPV-31 (12.2%)

Table 1 Prevalence of HPV infection by histologic classification (n = 86). Histology

Normal (n = 13) Cervicitis (n = 26) Normal/cervicitis (n = 39) All lesions (n = 47) CIN 1 (n = 20) CIN 2/3 or CIS (n = 23) ICC (n = 4)

HPV statusa Negative

Positive

8 (61.5) 11 (42.3) 19 (48.7) 6 (12.8) 5 (25.0) 1 (4.3) 0 (0.0)

5 (38.5) 15 (57.7) 20 (51.3) 41 (87.2) 15 (75.0) 22 (95.7) 4 (100.0)

OR (95% CI)

P value

Reference 2.18 (0.56–8.51) Reference 6.49 (2.24–18.8) 2.85 (0.87–9.38) 20.9 (2.56–171) NC

NA 0.257 NA b 0.001 0.079 b 0.001 0.086

Abbreviations: CI, confidence interval; CIN, cervical intraepithelial neoplasia; CIS, carcinoma in situ; ICC, invasive cervical carcinoma; NA, not applicable; NC, not computable; OR, odds ratio. a Values are given as number (percentage) unless otherwise indicated.

J. Ribeiro et al. / International Journal of Gynecology and Obstetrics 125 (2014) 107–110 Table 2 Distribution of HPV genotypes (n = 49).a HPV genotype

No. (%)

HR HPV, single infection All HR genotypes 16 18 31 33 39 52 56 58 59 pHR HPV, single infection All pHR genotypes 53 66 82 LR HPV, single infection All LR genotypes 11 61 70 Co-infection All samples with co-infection 16 + 83 39 + 53

40 (81.6) 20 (40.8) 1 (2.0) 6 (12.2) 2 (4.1) 3 (6.1) 1 (2.0) 1 (2.0) 5 (10.2) 1 (2.0) 4 (8.2) 1 (2.0) 2 (4.1) 1 (2.0) 3 (6.1) 1 (2.0) 1 (2.0) 1 (2.0) 2 (4.1) 1 (2.0) 1 (2.0)

Abbreviations: HPV, human papillomavirus; HR, high risk; LR, low risk; pHR, probable high risk. a Results were inconclusive for 4 additional HPV-positive samples.

and HPV-58 (10.2%); co-infections were rare (n = 2 [4.1%]) and involved at least 1 high-risk HPV type (Table 2). The physical status of HPV DNA was characterized in 19/21 HPV-16positive samples. Overall, 6/19 (31.6%) samples had integrated HPV-16 DNA; 1 sample had integrated DNA only and 5 samples had both integrated and episomal HPV-16 DNA. The prevalence of HPV-16 DNA integration was 0/1 (0.0%) for cervicitis, 2/5 (40.0%) for CIN 1, 0/2 (0%) for CIN 2, 2/6 (33.3%) for CIN 3, 1/1 (100.0%) for CIS, and 1/4 (25.0%) for ICC (data not shown). The frequency of HPV-16 integration into the host genome did not differ significantly by histologic classification (P = 0.578) or patient age (P = 0.440) (data not shown). 4. Discussion Persistent infection with 1 or more carcinogenic types of HPV is considered to be crucial for the development of intraepithelial lesions of the cervix that may progress to high-grade dysplasia or ICC [3]. Controlling HPV infection is thought to be the key to eradicating cervical cancer, and HPV vaccination promises to be an effective preventive measure that could help to reduce the global burden of cervical cancer within 20 years [19]. Hence, it is important to conduct studies that characterize HPV infection among specific populations, not only to collect epidemiologic data but also to improve screening programs, to develop future

Table 3 Characterization of human papillomavirus genotypes in the study population (n = 53).a Histology

Low-risk

High-risk

Probable high-risk

Inconclusive

Normal (n = 3) Cervicitis (n = 13) CIN 1 (n = 13) CIN 2 (n = 5) CIN 3 (n = 12) CIS (n = 3) ICC (n = 4) Total (n = 53)

0 (0.0) 2 (15.4) 0 (0.0) 1 (20.0) 0 (0.0) 0 (0.0) 0 (0.0) 3 (5.7)

2 (66.7) 7 (53.8) 13 (100.0) 3 (60.0) 11 (91.7) 2 (66.7) 4 (100.0) 42 (79.2)

1 (33.3) 1 (7.7) 0 (0.0) 1 (20.0) 1 (8.3) 0 (0.0) 0 (0.0) 4 (7.5)

0 (0.0) 3 (23.1) 0 (0.0) 0 (0.0) 0 (0.0) 1 (33.3) 0 (0.0) 4 (7.5)

Abbreviations: ICC, invasive cervical carcinoma; CIN, cervical intraepithelial neoplasia; CIS, carcinoma in situ. a Values are given as number (percentage).

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clinical approaches based on type-specific strategies, and to assess the impact and cost-effectiveness of HPV vaccines. The present study was performed on samples from 88 women who attended the Gynecology Service at the study institution because they had a previous history of cancer. The resulting bias in population sampling can be considered to be a limitation of the study. Nevertheless, the study provides important data. A surprising finding was that 51.3% of women without a clinical lesion tested positive for HPV infection; this included 38.5% of women with normal samples and 57.7% of women with cervicitis. This frequency is higher than that described in other studies [7,8,13], where the frequency of HPV infection among asymptomatic women varied from 10% to 26%. A possible explanation for this discrepancy could be that the present study was a hospital-based study, and some of the women without a clinical lesion might have been treated for cervical neoplasia in the past. The prevalence of HPV infection among women with intraepithelial lesions, including carcinomas, was similar to that described in worldwide studies [6,11,12,20,21]. To improve the clinical utility of HPV detection, several methods have been developed to determine the HPV genotype. We used the method described by Nobre et al. [17], which allows good discrimination between mucosal HPV types, including several HPV types that are underdiagnosed by the most common diagnostic methods. In the present study, high-risk HPV types were the most common genotypes, with HPV-16 and HPV-31 having the highest frequencies. In fact, HPV-16 has been described as the most common type among European women, followed by HPV-31 and HPV-58 [7,11–14]. The second most common high-risk type associated with cervical cancer is HPV-18. However, this type it was not among the most frequent genotypes in the present population. Other data from Portuguese populations indicate that HPV18 is a frequent genotype in invasive cancers, whereas it is rare in preneoplastic lesions [13,15]. In the present study, the frequency of HPV-18 might have been low because the study included very few ICC cases compared with a relatively large number of preneoplastic lesions. Other common genotypes include HPV-53 and HPV-66, which are currently underdiagnosed, and there is a debate among the scientific community whether they should be considered as probable high-risk types or as high-risk types [7,11–14,22]. The MY09/11 PCR primers amplify some HPV genotypes with less efficiency [17]. This could explain the difficulty in identifying the HPV genotype in 4 samples. In addition to genotype considerations, some authors [9,10,23] have discussed the importance of viral integration as an indicator of malignant progression in cervical cancer. In the present study, the majority (13/19) of women without a cervical lesion had episomal HPV-16 DNA only, whereas HPV-16 DNA integration was common for most lesion types, in agreement with previous results [18,24]. Although the association between cervical histology and integration of HPV DNA into the host genome was not statistically significant in the present study, viral integration is probably an early event in cervical carcinogenesis because it already occurred in low-grade lesions. The presence of integrated HPV-16 DNA in normal cervical epithelial cells and in lowgrade lesions has also been described in another study [25]. However, the findings must be confirmed in a larger study because the number of samples with integrated DNA in the present study was small. Such research will help to confirm whether women with integrated HPV DNA would benefit from closer follow-up. In addition, information on the physical status of other HPV genotypes is required. The present finding that only 1/4 invasive carcinomas had an integrated HPV-16 genome might be explained by methodologic limitations: real-time PCR may produce false-negative results for HPV-16 integration if the quantity of integrated DNA is low and that of episomal DNA is high, and the extent of HPV-16 integration might have been underestimated if HPV-16 disruption occurred outside the E2 region. Although the present study was performed with a small number of samples, the results indicate that HPV-16 and -31 are the most common genotypes in the Portuguese population. The existence of genotypes

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other than HPV-16 and -18 among Portuguese women may reduce the impact of HPV vaccination on cervical cancer prevention. The present findings also indicate that HPV-16 integration might be an early event in cervical carcinogenesis. Acknowledgments The Portuguese League Against Cancer (Liga Portuguesa Contra o Cancro – Núcleo Regional do Norte) provided a grant to J.R. Conflict of interest The authors have no conflicts of interest. References [1] zur Hausen H. Human papillomavirus & cervical cancer. Indian J Med Res 2009;130(3):209. [2] Zur Hausen H, Rösl F. Pathogenesis of cancer of the cervix. Cold Spring Harb Symp Quant Biol 1994;59:623–8. [3] Woodman CB, Collins SI, Young LS. The natural history of cervical HPV infection: unresolved issues. Nat Rev Cancer 2007;7(1):11–22. [4] Salmerón J, Lazcano-Ponce E, Lorincz A, Hernández M, Hernández P, Leyva A, et al. Comparison of HPV-based assays with Papanicolaou smears for cervical cancer screening in Morelos State, Mexico. Cancer Causes Control 2003;14(6):505–12. [5] Villa LL. Prophylactic HPV, vaccines: reducing the burden of HPV-related diseases. Vaccine 2006;24(Suppl. 1):S23–8. [6] de Sanjose S, Quint WG, Alemany L, Geraets DT, Klaustermeier JE, Lloveras B, et al. Human papillomavirus genotype attribution in invasive cervical cancer: a retrospective cross-sectional worldwide study. Lancet Oncol 2010;11(11):1048–56. [7] de Sanjosé S, Diaz M, Castellsagué X, Clifford G, Bruni L, Muñoz N, et al. Worldwide prevalence and genotype distribution of cervical human papillomavirus DNA in women with normal cytology: a meta-analysis. Lancet Infect Dis 2007;7(7):453–9. [8] Bruni L, Diaz M, Castellsagué X, Ferrer E, Bosch FX, de Sanjosé S. Cervical human papillomavirus prevalence in 5 continents: meta-analysis of 1 million women with normal cytological findings. J Infect Dis 2010;202(12):1789–99. [9] Pett M, Coleman N. Integration of high-risk human papillomavirus: a key event in cervical carcinogenesis? J Pathol 2007;212(4):356–67. [10] Ramanakumar AV, Goncalves O, Richardson H, Tellier P, Ferenczy A, Coutlée F, et al. Human papillomavirus (HPV) types 16, 18, 31, 45 DNA loads and HPV-16 integration in persistent and transient infections in young women. BMC Infect Dis 2010;10:326.

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Characterization of human papillomavirus genotypes and HPV-16 physical status in cervical neoplasias of women from northern Portugal.

To determine human papillomavirus (HPV) genotypes and the physical status of HPV-16 DNA among women from northern Portugal with cervical lesions...
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