Genotyping of human papillomavirus in cervical intraepithelial neoplasia in a high-risk population†
Carmen G. Ili and Priscilla Brebi contributed equally to this work.
Abstract
Infection with the human papillomavirus (HPV) is responsible for 99.7% of cervical cancers, the second most prevalent neoplasia in women worldwide and the fifth leading cause of death by cancer in this population. In Chile, the incidence rate is 14.4 cases per 100,000 women per year and it is considered a significant public health problem. The natural history of cervical cancer begins gradually from low-grade and high-grade squamous intraepithelial lesions to an invasive disease. In this study the frequency of HPV types was determined by HPV genotyping with reverse line blot hybridization in 200 cytobrushes of women with preneoplastic lesions in a high-risk population. HPV DNA was found in 89% of the lesions (83.3% of low-grade squamous intraepithelial lesions and 93.6% of high-grade squamous intraepithelial lesions). Multiple HPV infections were found in 14.4% and 15.5% of low- and high-grade lesions, respectively. HPV 16 was the most frequent genotype in single infections, followed by HPV 18. These results show that most of the preneoplastic lesions of the cervix (60%) were associated with HPV 16 and/or HPV 18, supporting the implementation of an HPV vaccination program in this high-risk population. J. Med. Virol. 83:833–837, 2011. © 2011 Wiley-Liss, Inc.
INTRODUCTION
Human papillomavirus (HPV) is the most important etiological factor for cervical cancer and its precursor lesions [Walboomers et al., 1999]. Cervical cancer is the second most prevalent neoplasia [Clifford et al., 2003b] and the third leading cause of death in women worldwide [Ferlay et al., 2010]. In Chile, cervical cancer is the sixth cause of death from malignant tumors in women [WHO/ICO, 2010] and the most frequent cause of death by carcinoma in women between 35 and 54 years [Ferreccio et al., 2004; WHO/ICO, 2010]. About 99.7% of cases of cervical cancers are attributed to HPV infection [Clifford et al., 2003a], which is considered to be the most frequently sexually transmitted disease in the world [Woodman et al., 2007].
Precancerous lesions of the cervix can be differentiated into low-grade squamous intraepithelial lesions and high-grade squamous intraepithelial lesions [Burd, 2003]. HPV detection is considered to be a complement to the clinical diagnosis of patients affected with these types of lesions [Lai et al., 2006]. HPV is present in different percentages in precancerous lesions: it is found between 59% and 82% of low-grade squamous intraepithelial lesions, while in high-grade squamous intraepithelial lesions the frequency is generally over 80% [Kulasingam et al., 2002, 2006; Clifford et al., 2003a; Huang et al., 2006].
More than 200 genotypes of HPV have been described, but only 40 are related to the anogenital mucosa. These HPV types have been divided into two groups according to the oncogenic risk. Low oncogenic risks HPV are 6, 11, 40, 42, 43, 44, 54, 61, 70, 72, and 81. HPV 6 and 11 are the most frequent types found in genital warts. High oncogenic risk HPV are 16, 18, 31, 33, 34, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 67, 68, 73, and 82. HPV 16 and 18 are the most frequent types found in cervical carcinomas [Munoz et al., 2003]. These two genotypes have been found to be responsible for 70% of all invasive cervical cancers worldwide [Bosch et al., 1995; Munoz et al., 2003].
The prevalence of HPV in Chile has been described in a limited number of studies, in normal samples [Ferreccio et al., 2004; Clifford et al., 2005a], in a few samples of precancerous lesions [Melo et al., 2003; Aedo et al., 2007], and in cervical cancer [Bosch et al., 1995; Melo et al., 2003; Roa et al., 2009; Valdivia et al., 2010]. The present study illustrates the prevalence of HPV in a large number of precancerous lesions found in a high-risk population group, due to the importance of finding the principal HPV genotypes infecting preneoplastic lesions in the Chilean population, in order to evaluate the future implementation of a vaccination program in this country.
MATERIALS AND METHODS
Collection of Clinical Samples
A total of 200 cytobrush samples from cervical scrapes were analyzed, the histological diagnoses of which were confirmed by biopsy. Of these samples, 90 were low-grade squamous intraepithelial lesions and 110 were high-grade squamous intraepithelial lesions. The samples were collected at the Doctor Hernán Henríquez Aravena Hospital, in Temuco, Chile from 2004 to 2008. The patients signed an informed consent before the samples were taken. This study was approved by Ethics Committees of the Hernán Henríquez Aravena Hospital and the Universidad de La Frontera's Faculty of Medicine.
Genotyping
The cytobrush samples were deposited in tubes with lysis buffer for DNA extraction. The DNA obtained underwent an integrity control, based on the amplification of a fragment (268 bp) of the beta globin gene using GH20 and PCO4 primers and PCR conditions described previously [Aedo et al., 2007]. Amplification of the L1 fragment for HPV detection was performed using GP5+ and biotin GP6+ primers as described previously [van den Brule et al., 2002]. Reverse line blot analysis was performed on the same samples to ascertain the HPV genotype as described [van den Brule et al., 2002]. Briefly, modified oligoprobes (18 oncogenic and 18 non-oncogenic HPVs) were used for the analysis [van den Brule et al., 2002]. Oligoprobes bound covalently to a membrane (Biodyne C; Pall Bio-Support West Chester, OH) were activated with EDAC 16% (w/v) (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, Sigma, St. Louis, MO), using the Miniblotter system (MN 45; Immunetics, Boston, MA). The PCR GP5+/bioGP6+ products were denaturalized at 96°C and cooled in ice prior to the hybridization process. These products were added to each Miniblotter channel, perpendicularly to the oligoprobe lines. Hybridization was performed after 1 hr and the membrane was removed and washed. Subsequently, the membrane was incubated with streptavidin–peroxidase conjugate (Roche, Basel, Switzerland), washed and incubated with ECL fluid (Amersham Biosciences, Piscataway, NJ), exposed to a film (Hyperfilm; Amersham Biosciences), and developed using standard reagents to detect the hybridization signal.
Control Samples
A panel of 36 HPV viral types was used for positive controls. HPV 16, 18, 31, and 33 corresponded to commercial plasmid clones (ATCC) and the remaining HPV types were provided by Dr. Peter Snijders (VU University Medical Center, Amsterdam, The Netherlands). Negative controls consisted of commercial genomic DNA (Promega, Madison, WI) and deionized water.
Definitions for Single and Multiple HPV Infections
A single HPV infection was defined as the detection of only one positive HPV genotype signal in the Reverse Line Blot. A multiple infection was defined as the presence of a positive signal for 2 or more genotypes. In multiple infections, the predominant type was recorded, meaning the HPV type presenting the strongest signal.
RESULTS
HPV genotyping was performed on 200 samples, 90 of which (45%) were low-grade squamous intraepithelial lesions and 110 (55%) high-grade squamous intraepithelial lesions. The age range of low-grade squamous intraepithelial lesions patients was between 18 and 70 years, with a mean of 44 years: 15.6% were aged between 18 and 30 years; 23.3% between 31 and 40 years; 34.4% between 41 and 50 years; and 26.7% over 50 years. The age range of high-grade squamous intraepithelial lesions patients was between 20 and 77 years, with a mean of 37 years: 40.9% were aged between 18 and 30 years; 30.0% between 31 and 40 years; 15.5% between 41 and 50 years; and 13.6% over 50 years.
HPV Detection
All samples were found to have good DNA integrity by beta globin PCR. L1 viral gene PCR was performed followed by reverse line blot technique on all samples. These analyses produced 178 (89%) positive samples for HPV.
HPV Typing in Low-Grade Squamous Intraepithelial Lesions
HPV was present in 83.3% of low-grade squamous intraepithelial lesions; 68.9% in single infections and 14.4% in multiple infections. For single infections, HPV 16 was the most frequent genotype (41.1%). HPV 11 (7.8%), HPV 18 (4.4%), and HPV 31 (3.3%) were found at lower frequencies, followed by HPV 33, 51, 52, and 56 each with 2.2% of the total and HPV 6, 45, and 66 each of them with 1.1% of the total (Table I). Of all the genotypes found in this kind of lesions, 84.6% were in the high-risk HPV group and only 15.4% were in the low-risk group. A 14.4% of the samples were infected with two or more types of HPV. HPV 16 and 18 were the most frequent types found in multiple infections (8.9%), together or in combination with one of the following types: 11, 42, 45, 52, 58, 66, 70, and 72. HPV 16 and/or 18 were involved in 60% of low-grade squamous intraepithelial cases. HPV 26, 34, 35, 39, 40, 42, 43, 44, 53, 54, 55, 57, 59, 61, 68, 71, 72, 73, 81, 82/IS39, 82/MM4, 83, 84, and CP6108 viral genotypes were not found in low-grade squamous intraepithelial lesions.
| Type of HPV infection | n | Percentage of HPV type | |
|---|---|---|---|
| Over total number of cases | Over total number of SI or MI cases | ||
| HPV-negative cases | 15 | 16.7 | — |
| Single infections (SI) | 62 | 68.9 | 100.0 |
| HPV 16 | 37 | 41.1 | 59.7 |
| HPV 11 | 7 | 7.8 | 11.3 |
| HPV 18 | 4 | 4.4 | 6.5 |
| HPV 31 | 3 | 3.3 | 4.8 |
| HPV 33 | 2 | 2.2 | 3.2 |
| HPV 51 | 2 | 2.2 | 3.2 |
| HPV 52 | 2 | 2.2 | 3.2 |
| HPV 56 | 2 | 2.2 | 3.2 |
| HPV 6 | 1 | 1.1 | 1.6 |
| HPV 45 | 1 | 1.1 | 1.6 |
| HPV 66 | 1 | 1.1 | 1.6 |
| Multiple infections (MI) | 13 | 14.4 | 100.0 |
| HPV 16/18 | 8 | 8.9 | 61.5 |
| HPV 16/other | 3 | 3.3 | 23.1 |
| HPV 16/18/other | 1 | 1.1 | 7.7 |
| HPV 11/16/other | 1 | 1.1 | 7.7 |
| Total | 90 | 100 | — |
HPV Typing in High-Grade Squamous Intraepithelial Lesions
HPV was present in 93.7% of high-grade squamous intraepithelial lesions: 78.2% in single infections and 15.5% in multiple infections. For single infections, HPV 16 was the most frequent genotype (44.5%). HPV 18 (9.1%) was found at to a lesser extent, followed by HPV 31, 45, 51, 53, and 58 each of them with 2.7% of the total, HPV 35, 42, 56, 59, and 70 with each of them 1.8% of the total and HPV 33 and 67 each of them with a frequency of 0.9% of the total (Table II). Of all the genotypes found, 89.3% were in the high-risk HPV group and only 10.7% were in the low-risk group. A 15.5% of the samples were infected with two or more types of HPV. HPV 16 and 18 were the most frequent types found in multiple infections, together or in combination with one of the following types: HPV 33, 42, 44, 52, 56, 66, 68, and 81. In general, HPV 16 and/or 18 were involved in 65.5% of these types of lesion. The following viral genotypes were not found: HPV 6, 11, 26, 34, 39, 40, 43, 54, 55, 57, 61, 70, 71, 72, 73, 82/IS39, 82/MM4, 83, 84, and CP6108.
| Type of HPV infection | n | Percentage of HPV type | |
|---|---|---|---|
| Over total number of cases | Over total number of SI or MI cases | ||
| HPV-negative cases | 7 | 6.4 | — |
| Single infections (SI) | 86 | 78.2 | 100.0 |
| HPV 16 | 49 | 44.5 | 57.0 |
| HPV 18 | 10 | 9.1 | 11.6 |
| HPV 31 | 3 | 2.7 | 3.5 |
| HPV 45 | 3 | 2.7 | 3.5 |
| HPV 51 | 3 | 2.7 | 3.5 |
| HPV 53 | 3 | 2.7 | 3.5 |
| HPV 58 | 3 | 2.7 | 3.5 |
| HPV 35 | 2 | 1.8 | 2.3 |
| HPV 42 | 2 | 1.8 | 2.3 |
| HPV 56 | 2 | 1.8 | 2.3 |
| HPV 59 | 2 | 1.8 | 2.3 |
| HPV 70 | 2 | 1.8 | 2.3 |
| HPV 33 | 1 | 0.9 | 1.2 |
| HPV 67 | 1 | 0.9 | 1.2 |
| Multiple infections (MI) | 17 | 15.5 | 100.0 |
| HPV 16/other | 6 | 5.5 | 35.3 |
| HPV 16/18 | 5 | 4.5 | 29.4 |
| HPV others | 4 | 3.6 | 23.5 |
| HPV 16/18/other | 1 | 0.9 | 5.9 |
| HPV 18/other | 1 | 0.9 | 5.9 |
| Total | 110 | 100 | — |
DISCUSSION
It is known that HPV infection is the principal etiological factor for cervical cancer. Several studies on both tissue biopsy or cytobrush samples have shown that HPV DNA is present in a high percentage of cases. HPV is found in 99.7% of cervical cancer biopsies [Motoyama et al., 2004]. Therefore, it is important to determine the genotypes of HPV involved in each population, and particularly in intraepithelial lesions, in order to take prophylactic measures against cervical cancer. Knowledge of the distribution of HPV genotypes in women will make it possible to design new diagnostic techniques.
In the present study, cytobrush samples were used. Cytobrush is a fresh cervical cell suspension without formalin or other fixative which could induce DNA fragmentation [Melo et al., 2005]. Cytobrush samples have excellent DNA yield, allow for good HPV genotyping, and are not invasive.
The prevalence of HPV was 83.3% in low-grade squamous intraepithelial lesions and 93.7% in high-grade squamous intraepithelial lesions. This is higher than in other studies, where 71.1% and 84.2% of cases were found to be HPV positive in both kind of precancerous lesions, respectively [Clifford et al., 2003a, 2005b]. Both of these studies used mainly biopsy samples, which may explain why the prevalence of HPV was greater in the present study. When cytobrush is used to detect HPV in invasive cervical cancer, positivity rates are between 90 and 100% in all cases [Walboomers et al., 1999].
The most frequent HPV genotype found in both kinds of lesions was HPV 16, which is known to be the most frequent genotype worldwide, is usually detected in half of cervical cancer cases. HPV 16 is also found at lower, but nonetheless significant rates in preneoplastic lesions, and even in normal cervical samples [Clifford et al., 2003a,b, 2005b; Ferreccio et al., 2004; Roa et al., 2009]. Additional important genotypes detected were HPV 18 and 31 (both highly oncogenic), which showed a similar result to those of cervical cancer studies in Latin America and Chile [Munoz et al., 2003; Roa et al., 2009]. The percentage of multiple infections found in preneoplastic lesions is higher than what is generally found in others studies perform in cancer samples; however, this result could be related to the high sensitivity of the reverse line blot assay and the quality of the samples.
An important finding is that HPV 6 and 11, both low-risks HPV, were present only in low-grade squamous intraepithelial lesions. In fact, in high-grade squamous intraepithelial lesions the low-risk HPV were present only in multiple infections in the presence of a high risk HPV, thereby supporting the current paradigm that only high-risk HPVs is associated with cancer, while low-risk HPVs are cleared by the immune system [Munoz et al., 2003].
The distribution of HPV types in the Chilean population is similar to that in other regions of Latin America, with HPV 16 and HPV 18 found to be the most frequent genotypes. In low-grade squamous intraepithelial lesions, HPV 16 and HPV 18 infections together correspond to 60% of the total of HPV positive cases for these lesions, against 65.5% for high-grade squamous intraepithelial lesions. Chile has a cervical cancer incidence of 14.4 per 100,000 women [WHO/ICO, 2010]. The implementation of HPV vaccines may protect up to 60–65.5% of the HPV infections which cause intraepithelial neoplasias in this country and on the whole the incidence rates of cervical cancer could be decreased with PAP screening in the future.
The reverse line blot technique for HPV detection and typing was successful because it identified the viral genotypes considered important epidemiologically, showing their distribution and frequency. A large number of samples can be tested at the same time with 38 specific oligoprobes for HPV.
In conclusion, the frequency of HPV found in intraepithelial lesions was higher than published previously, basically due to the good quality of the samples and the high performance of the reverse line blot technique, which is sufficiently sensitive to detect only a few copies of HPV, making of this procedure a good complement to the diagnosis and prognosis of patients with cervical preneoplastic lesions. Furthermore, the most frequent HPV genotypes in Chile could be prevented by the implementation of a vaccination program.
