Cervical cancer is the second most common cancer in women worldwide; the annual incidence is estimated at 500,000, with 274,000 fatalities.1 In recent years, a number of studies have reported that human papillomavirus (HPV) infection is the major causative factor in the development of cervical cancer, with HPV DNA present in 99.7%. In Thailand, cervical cancer is one of the most common types of cancer, with age-standardized incidence rates of 24.5 per 100,000 women. The incidence of cervical cancer has been recorded at approximately 9,999 new cases per annum.2 More than 100 HPV genotypes have been identified, with 40 commonly infecting the anogenital epithelium and 15 thought to be carcinogenic.3 Thus, these viruses have been classified as low- and high-risk types depending on their propensity to cause cancer.4,5
The implementation of HPV vaccines will be the future trend of primary prevention for cervical cancer.6–10 Awareness of HPV genotype distribution will form the basis of guidelines for HPV-based cervical cancer screening and cost-effective prophylactic HPV vaccine policy and assessment of the effect of vaccination on HPV infection in each geographic area.6 At present, 2 prophylactic vaccines against HPV infection have been licensed for clinical use. Human papillomavirus vaccination is part of the national immunization program of many countries. Both vaccines (Cervarix from GlaxoSmithKline, Middlesex, UK; and Gardasil from Merck, KGaA and Darmstadt, Germany) contain viruslike particles derived from 2 high-risk types (HPV 16 and 18). Gardasil, in addition, covers 2 low-risk types (HPV 6 and 11) that cause benign genital warts. Both vaccines are proven to be highly effective in preventing the development of cervical neoplasia caused by the targeted HPV types.11–14 These 2 vaccine-covered high-risk HPV types (HPV 16 and 18) account for 70% of invasive cervical cancers and 52% of high-grade cervical intraepithelial neoplasia worldwide, but geographical variations have been observed.15–17 As future vaccine candidates18 are likely to be HPV genotype specific, awareness of HPV genotype distribution in lesions detected by routine screening also helps predict the effect of vaccination on the reduction of abnormal findings in cervical screening programs.
Over the last few years, several groups have searched for an effective genotyping test for HPV. Our previous study,19 comparing HPV genotyping methods by direct sequencing and a line probe assay (INNO-LiPA) in Thai women, found that both methods have advantages and disadvantages. Hence, we should select the method most suitable for the study objective, budget, and predominance of HPV genotype in any given area. INNO-LiPA is good for detecting multiple HPV infection, but not all HPV genotypes can be typed. Moreover, the genotype kit sometimes does not allow discrimination between as many genotypes as indicated in the interpretation chart. In the present study, we decided to determine the prevalence of HPV genotypes in cervical cancer specimens in Thailand by applying the INNO-LiPA method. Unfortunately, owing to the nondiscrimination problem previously mentioned, DNA chip method was used for genotypic confirmation. The aim of this study was to determine the distribution and the prevalence of HPV genotypes in a group of women with a diagnosis of cervical cancer in Thailand, from which no more prior information has been available by means of the combined use of INNO-LiPA and DNA chip method. The information thus obtained will show the distribution of HPV genotypes and which genotypes are most prevalent in cervical cancer in Thailand in comparison with other geographical areas. Moreover, it will indicate whether future application of HPV vaccines will be successful.
MATERIALS AND METHODS
Study Population and Clinical Specimens
The Ethics Committee of the hospital and Faculty of Medicine, Chulalongkorn University, approved the study protocol. All the studied specimens were anonymous with a coding number for analysis, and permission was granted by the director of the hospital. The samples included in this study comprised 155 cervical cancer specimens of Thai women mainly from central Thailand obtained from biopsy tissue at the National Cancer Institute, Thailand, from December 2010 to May 2011. Cytological and International Federation of Gynecology and Obstetrics (FIGO) stage data were classified following the International Agency for Research on Cancer criteria.20 All specimens were collected in phosphate-buffered saline and stored at −70°C until used.
DNA was obtained by standard organic extraction (phenol-chloroform) and alcohol precipitation of the specimens. Briefly, tissues were lysed in 400 mL of lysis buffer (10-mmol/L Tris-HCl, pH 8.0; 0.1-mol/L EDTA, pH 8.0, 0.5% sodium dodecyl sulfate). Samples were incubated at 95°C for 30 minutes, mixed for 2 minutes, and digested with 50 µL of proteinase K (Amresco, Solon, OH) (20 mg/mL) at 50°C overnight, then heated to 95°C for 10 minutes to inactivate proteinase K. Consequently, the phenol-chloroform extraction was performed followed by high-salt isopropanol precipitation as described previously.21 The purified material was resuspended in a final volume of 30 µL of deionized water.
HPV DNA Amplification
Human papillomavirus DNA was amplified by using the INNO-LiPA HPV genotyping extra amp kit (Innogenetics N.V., Ghent, Belgium), following the manufacturer’s instructions. The SPF10 primer set used in the INNO-LiPA HPV genotyping extra amplifies a 65-base pair region in the L1 open reading frame and has the potential to amplify at least 54 HPV types. A multi-amplified biotinylated target sequence is obtained after 40 cycles of polymerase chain reaction.
One-hundred forty-nine HPV-positive DNA samples were genotyped by INNO-LiPA HPV genotyping Extra (Innogenetics N.V.) following the manufacturer’s instructions. This kit has been designed for the identification of 28 different genotypes of HPV by detection of specific sequences in the L1 region of the HPV genome. The assay covers all currently known high-risk HPV genotypes and probable high-risk HPV genotypes (16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, 73, and 82) as well as a number of low-risk HPV genotypes (6, 11, 40, 43, 44, 54, and 70) and some additional types (69, 71, and 74). Amplified products were denatured under alkaline conditions and immediately incubated with the test strip in hybridization buffer. The hybridization patterns were interpreted by a fully automated scanner and Line Reader and Analysis Software for LiPA HPV software. This genotyping kit does not allow discrimination between many genotypes such as HPV 18 and HPV 18 and 39, HPV 58 and HPV 58 and 52, and the like as indicated in the interpretation chart. The samples showing nondiscriminated genotypes mentioned in the chart were subjected to the DNA chip method to confirm their HPV genotypes.
Electrochemical DNA Chip
The loop-mediated isothermal amplification reaction and detection were performed using electrochemical DNA chip (Toshiba Company, Tokyo, Japan) according to the manufacturer’s instructions. Human papillomavirus genotyping was accomplished by hybridization and subsequent detection by electrochemical DNA chip. The electrochemical DNA chip contained specific DNA probes in the L1 region of 13 high-risk HPVs (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68). The loop-mediated isothermal amplification conditions were denaturation at 95°C for 5 minutes, followed by 90 minutes at 65°C and 5 minutes at 80°C. The automated hybridization was performed using a GenelyzerTM for Medical Field GLH-2C601 (Toshiba Company, Tokyo, Japan). The results are shown in the bar chart between electric current peak values on the y-axis and HR-HPV genotypes on the x-axis.
All data of the HPV genotype-specific prevalence was analyzed, calculated, and presented as percentage.
The clinical characteristics and HPV genotyping of the study patients are summarized in Table 1. A total of 155 cervical cancer specimens collected from patients at the National Cancer Institute between December 2010 and May 2011 were enrolled in this study. After histological findings, clinical lesions, physical examination, and radiological input, 148 samples could be grouped into FIGO stage I to stage IV cervical cancer; 7 samples were not determined by histology and thus were indicated as not determined. The ages of the 155 enrolled patients ranged from 26 to 83 years, with a mean age of 49.9 years. Most samples were from women aged 51 to 60 years (50/155 [32.3%]), and most samples were characterized as FIGO stage II and III cervical cancer with 32.3% and 29.7%, respectively. Nearly all patients (94.2%) used to have sexual intercourse. Of the patients, 27.7% had a family history of cancer. Interestingly, 30.2% of this group had cancers affecting the urogenital tract.
We performed HPV genotyping by using the INNO-LiPA method. Six specimens were negative for HPV DNA as shown in Table 1. When the INNO-LiPA method was used alone, some specimens had a problem about the overlap line and could not be typed genotypically. Therefore, we confirmed the genotype of these specimens by using the DNA chip method. Single HPV genotype infections (129/155 [83.2%]) were more common than multiple infections (18/155 [11.6%]) as shown in Table 1. Multiple infections comprising double and triple or more genotypes amounted to 8.4% and 3.2%, respectively, as shown in Figure 1 and Table 2. Human papillomavirus 16/52 were the most common double types (4/13 [30.8%]). In this study, HPV genotyping by the INNO-LiPA method showed untypable genotypes in 2 specimens. All HPV genotypes found in this study could be classified as 13 high-risk HPV (HR-HPV: 16, 18, 33, 35, 39, 45, 51, 52, 53, 56, 58, 68, and 82), 2 low-risk HPV (44 and 70), and 2 additional types (HPV 74 and 69/71). The low-risk and additional types of HPV were detected in a part of multiple infections. Of the specimens, 94.8% had at least one HR-HPV genotype infection (data not shown).
The most frequent genotypes by decreasing order of frequency were as follows: HPV 16 (79/155 [51%]) followed by HPV 18 (31/155 [20%]), HPV 52 (16/155 [10.3%]), HPV 58 (9/155 5.8%]), and HPV 33 (7/155 [4.5%]). All genotypes except for genotype 52 were more frequently found as single as opposed to multiple infections (Fig. 2).
The distribution of HPV genotypes in the different FIGO stage I to stage IV cervical cancer samples is shown in Figure 3. Single infection was most frequently found in all stages. Among single infections in all stages, HPV16 was the most frequent genotype found in stages I to IV and also not determined at approximately 43.8%, 53.3%, 54.3%, 72.7%, and 66.7%, respectively.
The present study has shown the prevalence of HPV genotypes in cervical cancer of Thai women based on the INNO-LiPA and DNA chip methods. The 155 cervical cancer specimens were from Thai women aged between 26 to 83 years with a mean age of 49.9 years who were enrolled in this study. Upon performing INNO-LiPA for HPV DNA detection, 6 specimens were HPV DNA negative. This could imply non–HPV-related cervical cancer in 3.9% of the specimens or that the sensitivity of INNO-LiPA in HPV DNA detection in this study was approximately 96.1% (149/155). However, genotyping by the INNO-LiPA method may lead to a slightly overestimated prevalence rate owing to its higher clinical sensitivity compared to other methods. Compared to previous studies conducted on Thai women, this study described the prevalence of specific HPV genotype among cervical cancer cases mainly representative of the central parts of Thailand.22–34 When performing only the INNO-LiPA method for HPV genotyping, many genotypes could not be discriminated, for example, HPV 18 and HPV 18 and 39, HPV 58 and HPV 58 and 52, etc, as indicated in the interpretation chart; and thus, various specimens could not be identified. We decided to use the other genotyping method, the DNA chip method, which can detect 13 HR-HPV genotypes, to confirm the genotypes of these specimens. Upon combining both genotyping methods, all specimens could be analyzed correctly and specifically genotyped. We found that 94.8% of cervical cancer specimens had at least one HR-HPV genotype infection. Multiple HPV infection is less common than single HPV infection (11.6% vs 83.2%). This indicates that one HR-HPV genotype on its own is sufficiently virulent to cause cancer and thus might confirm that HPV is the causative agent for cervical cancer worldwide. The frequencies of multiple infections varied from 1.8% to 26.5%.35 The prevalence of multiple infections in the present study was lower than that from North Thailand (21.9%)28 but higher than that from South Thailand (3.7%).34
In the present study, we found that 83.2% of the cervical cancer specimens showed single infection, which is in agreement with the previous study by Lai et al,6 which found 82% of single infection. In contrast with our previous study19 on HPV genotyping performed on different groups of specimens that included not only cancer specimens but also normal, low-grade squamous intraepithelial lesion, and high-grade squamous intraepithelial lesion specimens, we found that in noncancer specimens, the percentage of specimens harboring single HPV type infection was quite equal to that containing multiple infections (44.2% vs 55.8%, respectively). This might indicate that during the initial stages of HPV infection, many types of HPV can be found, but once the cytology changes or progresses to the cancerous stage, a single HR HPV type can overwhelm the other genotypes and remain in the lesion on its own.
Single HR-HPV infection is quite common in all FIGO stages of cervical cancer. A previous study on prevalence of HPV DNA in women with normal cytology has established that worldwide, HPV prevalence was highest in women below the age of 35 and decreased in women of older age. In contrast with this study, which detected HPV DNA in women with a diagnosis of cervical cancer, most samples were from women aged 51 to 60 years, and most of them were characterized as FIGO stages II and III. Progression to advanced stage (stage II-III) of cervical cancer was more frequently found than the early stage (stage I) in women of older age (51–60 years), and the prevalence of cancer is quite low in the age group of younger than 30 and older than 60 years.
As for the worldwide distribution of HPV genotypes, HPV 16 is consistently the most prevalent type, whereas the frequencies of other genotypes vary in different geographic areas and ethnic groups.6,36 In our study, HPV 16 was the most common genotype, identified in 79 (51%) of the 155 cases, which is concordant with many other studies in Thailand and worldwide.6,23,28,31–33,36,37 Interestingly, the 5 most prevalent genotypes in this study were HPV 16, 18, 52, 58, and 33 by order of frequency as had also been observed by Clifford et al15 and Sanjose et al.36 The prevalence of HPV 52 and HPV 58 infection in this study was relatively high, culminating in the same results as previously reported.28 This finding supports the previous study in that HPV 52 has a larger impact on progression to cervical cancer in Thailand than elsewhere.28,31 In contrast to other genotypes, genotype 52 was found more frequently with multiple than single infections. The multiple infections found in this study comprised double, triple, quadruple, or more infections. The most common double infection was HPV 16/52. No coinfection by genotypes 16 and 18 was detected as they seem to be rather uncommon elsewhere also.28,31
The frequencies of HPV genotypes contributing to cervical cancer have been studied and reported elsewhere. The frequencies of HR-HPV genotype contributing to cervical cancer reported by our group were compared to those reported by Munoz et al37 and Siriaunkgul et al28 (Table 3). Based on our study, the most predominant genotype of HPV contributing cervical cancer worldwide was HPV 16. The frequencies of HPV DNA detection and the common double HPV infection from our study (approximately 90% and HPV 16/52, respectively) were similar to those of Thai women residing in the North.28 The efficacy of vaccines in preventing cervical cancer from HPV infection was approximately 70% (Table 3; prevalence of HPV 16 and HPV 18 were 51% and 20%, respectively). Human papillomavirus genotypes other than 16 and 18 causing carcinoma (account for 30%) found in this study are also shown in Table 3. All studies showed that HR-HPV genotypes have a potential risk to be an etiologic agent of cervical cancer by more than 90%.
Vaccination is a cost-effective method to prevent diseases caused by infectious agents. The most important aim of HPV vaccination is to reduce the incidence of cervical cancer and precancerous lesions. The other aim is to decrease the rate of cancers and other benign lesions related to HPV infection.38 In our study, the prevalence of genotypes 16 and 18, which are included in both the Merck and GlaxoSmithKline vaccines, amounted to approximately 71%. Thus, in light of the potential success of an HPV vaccination program in Thailand, either vaccine could prevent HPV-related cervical cancer in only approximately 70% of women. Although this vaccine has additional cross protection to HPV 31, 33, and 45,39 there are still other genotypes commonly detected in cervical cancer such as HPV 52, 58, and others. Therefore, screening programs such as Papanicolaou test, cytologic test, and HPV DNA detection in addition to educational and information programs are and will remain essential to prevent cervical cancer worldwide. Moreover, future generations of HPV vaccines should also include the other most common genotypes and decrease the severe adverse effects reported at the present time.
The authors thank the Higher Education Research Promotion and National Research University Project of Thailand, Office of the Higher Education Commission (HR1155A), Thailand Research Fund (DPG5480002), Research grant from the Faculty of Medicine, Srinakharinwirot University (336/2554), Toshiba Research and Development Center, The Commission on Higher Education, Ministry of Education, the Center of Excellence in Clinical Virology, Chulalongkorn University, CU Centenary Academic Development Project, and King Chulalongkorn Memorial Hospital for their generous support. The authors likewise thank the staff of the Department of Pathology, Samitivej Srinakharin Hospital, Thailand, and the Department of Obstetrics and Gynecology, National Cancer Institute for providing the samples. Finally, the authors thank Ms Petra Hirsch for reviewing the manuscript.
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