Abstract
Background. Human papillomavirus (HPV) infection is the major cause of cervical cancer, but the prevalence of different HPV types varies depending on geographical location and may change dramatically after introduction of HPV vaccination. Here, we aimed to gain some information regarding the recent prevalence of different HPV types, in cancer of the uterine cervix in the Stockholm region, before the introduction of public HPV vaccination in Sweden. Material and methods. From 215 diagnosed cervical cancer patients 2003–2008 at the Karolinska University Hospital, 160 pretreatment cervical cancer samples, including both squamous cell carcinomas (SCC) and adenocarcinomas (ADC) could be obtained. DNA was extracted from 154/160 of the SCC and ADC samples and assayed by Luminex Multiplex for 24 different HPV types, including 15 high-risk (HR), three putative HR and six low-risk types (LR). Results. We successfully analysed 154/215 (71.6%) of the locally diagnosed cases and found a high prevalence of HPV with 92.9% in all uterine cervix cancer cases, and 93.3% and 91.4 % in SCC and ADC, respectively. All HPV positive cases harboured HR types, either alone or as multiple infections. In SCC HPV16 dominated and together with HPV18 accounted for 69.7% of the cases, followed in prevalence by HPV33, 31 and 45. In ADC, HPV18 was more common than HPV16, and they were observed in all except one of the HPV positive samples. Conclusion. The prevalence of HPV16 and 18, followed by HPV33, 31 and 45 is high in SCC and ADC in the Stockholm region. Public HPV vaccination could potentially inhibit a large proportion of such tumours underlining the urgency to initiate HPV vaccination.
Worldwide, cancer of the uterine cervix is the third most common cancer among women, with over 529 000 new cases and about 275 000 deaths in 2008. However, there is a discrepancy in prevalence between developing countries, where >85% of all cervical cancer cases occur, and industrialised countries. Hence, the highest incidence is found in Eastern and Western Africa, while the lowest is found in Europe, Northern America and the Arabic countries. In Europe, cervical cancer is the seventh most common cancer in women, with over 54 000 new cases and 25 000 deaths annually [Citation1]. In the Nordic countries, an apparent reduction in the annual number of cases has been observed after the initiation of organised screening in the mid 1960s [Citation2]. However, up to 1300 new cases are diagnosed every year in the Nordic region and in Sweden about 450 new cases are reported annually [Citation3].
The vast majority of cervical cancer cases are squamous cell carcinomas (SCC), while adenocarcinomas (ADC) are somewhat less common. There are additionally several rare cancer types that are seldom diagnosed. In general, countries with good cervical cancer screening programs often report an increased proportion of ADC (∼25% in most Western countries) compared to unscreened populations [Citation4,Citation5].
The major cause of cervical cancer is persistent infection with different oncogenic human papillomavirus (HPV) types [Citation6]. Approximately 10% of all women are regarded to harbour HPV infection at a given time, and 83–99.7% of all cervical cancers have been reported to be associated with HPV infection [Citation7–11]. However, the prevalence of different HPV types varies somewhat in different regions of the world. HPV16 dominates worldwide and contributes to half of all cervical cancer, followed by HPV18, and together they contribute to around 70–76% of all invasive cervical cancers. HPV45, 31, 33, 58 and 52 frequently contribute to the remainder [Citation9–15].
There are currently two vaccines against HPV: Cervarix (GSK) and Gardasil (Merck). Both target the high-risk (HR) types HPV16 and 18. Gardasil additionally covers the low-risk (LR) types HPV6 and 11. In addition, both vaccines exhibit some cross protection against some other HPV types [Citation16–18]. In Sweden, a public vaccination programme of girls aged 10–12 is in the planning stages (originally planned for 2010). Although, there are studies on the distribution of HPV types in pre-cancerous lesions in Sweden, there are few studies, describing HPV types (especially covering more recent periods) in cervical cancer.
To gain information regarding the more recent prevalence of HPV types, before public HPV vaccination we retrospectively examined for HPV in cervical cancer (both SCC and ADC) at the Karolinska University Hospital, which covers more than half of the patients diagnosed in the Stockholm area during the period 2003–2008. The presence of HPV, as well as specific type, was analysed from formalin-fixed paraffin-embedded cervical cancer samples, using Luminex.
Material and methods
Patients and tumour samples
According to the Swedish Cancer Registry, between 2003 and 2008, in the County of Stockholm, 281 patients were diagnosed with squamous cell carcinomas (SCC) and 75 were diagnosed with adenocarcinomas (ADC). More than 60% of all patients with cervical cancer in the Stockholm region are diagnosed at the Department of Pathology and Cytology at the Karolinska University Hospital. The Department of Pathology and Cytology uses an electronic record, and all cases are classified according to the Systematised Nomenclature of Medicine (SNOMED) coding system. Searching the Department of Pathology and Cytology electronic database we identified 215 patients with cervical cancer between 2003–2008. Formalin-fixed paraffin-embedded tissue blocks containing representative tumour material was retrieved for 160 (74.4%) of these patients. The missing cases (55/215) were due to: a) the patient had advanced disease and did not go through surgery; b) insufficient sample material; c) inconsistently applied SNOMED coding. In total, 155/160 samples, i.e. 72.1% of the 215 cases were verified to contain sufficient tumour material for further analysis. Samples were macro-dissected in order to remove non-cancerous tissues. Only five samples with too little material were excluded for the analysis. One sample diagnosed as both SCC and ADC was excluded for further analysis, thus we covered 154/215 (71.6%) of the cases. The study was conducted according to permission 2008/813-31/2 from the Ethical Committee at Karolinska Institutet, Stockholm, Sweden.
Extraction of HPV DNA
Two 15 μm thick sections were cut from each paraffin-embedded sample. One paraffin control sample without any tissue was taken every 10th sample and treated in the same way to exclude possible cross-contamination. All tumours were macro-dissected to obtain as much tumour material as possible. In addition, during the DNA extraction one empty sample was added per five samples to assay for possible contamination during the DNA-preparation and PCR analysis. DNA was extracted according to the manufacturer's instructions for the High Pure RNA Paraffin Kit (Roche) without DNase treatment.
Multiplex HPV Genotyping by Luminex
A genotyping kit for HPV from Multimetrix (Heidelberg, Germany) was used for HPV typing. This included 24 HPV types: 15 HR types (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68, 73 and 82), three putative HR types (26, 53 and 66) and six LR types (6, 11, 42, 43, 44 and 70) classified according to Muñoz et al. [Citation19]. The samples were analysed in parallel as described by Schmitt et al. [Citation20]. The PCR amplication and hybridisation of PCR-products to Luminex beads, as well as the determination of cut-off values, were performed as previously described by Ramqvist et al. [Citation21].
Results
Patient age
According to the Swedish Cancer Registry, the median age for all 356 patients, e.g. patients diagnosed with squamous cell carcinoma (SCC) and patients diagnosed with adenocarcinoma (ADC) was 48 years. The median age of patients in our study with samples, including both the SCC and ADC groups, was 42 years. The age range of all patients was 22–95 years with 22–95 years in the SCC group and 25–91 years in the ADC group. The age range for patients where samples were available for analysis was 23–88 with 23–88 years in the SCC group and 25–81 years in the ADC group. Hence, patients with available samples were slightly younger than those who lacked samples.
Prevalence of different HPV types in cervix cancer including both SCC and ADC types
All 154 cervical cancer samples demonstrated PCR amplifiable DNA since they all tested positive for the presence of the beta-globin housekeeping gene by the Luminex multiplex assay. In total, 143/154 (92.9%) of the samples were HR HPV positive. The majority 131/154 (85.1%) were positive for only one HPV type, leaving 12/154 (7.8%) of the samples were positive for more than one HPV type ( and ). HPV16 and 18 dominated covering 90/154 (58.4%) and 29/154 (18.8%) of the samples. However, HPV16 and 18 occurred together in five cases, thus HPV16 and/or HPV18 accounted for 74.0% (114 cases) of all the samples. HPV33, 31 and 45 followed thereafter in prevalence accounting for 4.5%, 3.2% and 3.2% of the samples, respectively, covering 11.0% of the cases together. The remaining other analysed HR-HPV types (including HPV52) distributed equally in approximately one to four cases, with the exception of HPV58, which was not detected at all. When considering all infections with the HPV types (HPV16, 18, 31, 33, 45 and 52) that potentially may be covered by the present HPV vaccines, they accounted for 127/154 (82.5%) of the cases.
Table I. Distribution of different HPV types uterine cervical cancer identified by histotype and patient age.
Table II. Distribution of single and multiple HPV types in uterine cervical cancer identified by histotype.
The distribution of HPV types and age specific prevalence of HPV in SCC
In total, 111/119 (93.3%) SCC samples were infected by HPV. HPV16 and 18 were found in 76/119 (63.9%) and 12/119 (10.1%) of the SCC cases, respectively. However in five cases both HPV16 and 18 were present, hence HPV16 and 18 contributed to 83/119 cases (69.7%) ( and ). Single HPV infections without HPV16 or 18 were observed in 24/119 (20.2%) of the SCC cases; five cases each with HPV31 and HPV33, three with HPV45, two cases each with HPV51, HPV56 and HPV73 and finally one case each of HPV35, 39, 52, 59 and 68 (). Multiple infections without HPV16 or 18 were observed in the four remaining cases as shown in .
The distribution of HPV types and age specific prevalence of HPV in ADC
In total, 32/35 (91.4%) of the ADC samples were HPV positive (), of which all except one HPV45 positive case included either HPV16 or 18 (31/35, 88.6%) (). More specifically, 17 cases (48.6%) were HPV18 positive and 14 cases (40.0%) were HPV16 positive. In addition, one co-infection with HPV16 and 45 and one with HPV18 and 39 were observed, thus only 2/35 (5.7%) of the ADC samples were infected with more than one HPV type.
Discussion
In this study, we report a high prevalence of HPV (92.9%), and of HPV16 and/or HPV18 (74.0%) in pretreatment uterine cervix cancer samples, including squamous cell carcinoma (SCC) and adenocarcinoma (ADC), analysed from 71.6% of the patients diagnosed 2003–2008 at the Karolinska University Hospital in Stockholm. All HPV positive cases harboured HR HPV types, either alone (85.1%) or together with multiple infections (7.8%). HPV16 and 18 dominated and accounted for 58.4% and 18.8%, respectively, of the cases, and they were followed in prevalence by HPV33, 31 and 45. The only non-detected HR HPV type was HPV58 ().
We observed an overall high HPV prevalence (92.9%) in this study is similar to that reported globally and in Europe [Citation9,Citation12,Citation15]. In addition, our detection rate of both HPV16 (58.4%) and HPV18 (18.8%) was comparable with the prevalence range of HPV16 (52–67%) and HPV18 (14–20%) from data derived both globally and from other European countries [Citation9,Citation12,Citation15]. HPV16 was more prevalent in SCC samples, while HPV18 was more common in ADC (48.6%) as compared to in SCC (10.1%), which is in accordance to the findings of others [Citation9,Citation10,Citation12,Citation22]. HPV 31, 33 and 45 followed after HPV 16 and 18 in frequency, and accounted for 3.2%, 4.5% and 3.2% of the cases in this study, which is analogous to that reported in other European studies [Citation9,Citation10,Citation14,Citation15]. In addition, only a minority of the SCC (8.4%) and the ADC (5.7%) samples exhibited multiple HPV infections, which is similar to some, but not all other reports [Citation11,Citation13].
Both presently available HPV vaccines (Gardasil and Cervarix) are highly effective in preventing incident and persistent HPV16 and 18 infections [Citation16–18]. According to our data, infection or co-infection with HPV16 and 18 accounted for over 74.0% of the SSC and ADC cases. Infection with HPV types 31, 33, 45 and 52 (where there could be cross-protection by the present vaccines) without HPV16 and 18 accounted for another 11.7% of the cases. The data is in accordance to that of others and suggests that a large proportion of these tumours could be inhibited by future vaccination in the study area [Citation18]. Vaccination will likely change the distribution of HPV types in cervical cancer, and this information gives us an approximate baseline and could be useful for future for planning which next generation of HPV vaccines should be made available.
There are some limitations in our study. First the diagnosis time is limited to between 2003 and 2008, preventing the study of natural fluctuations in the prevalence of different HPV types over time. Nonetheless, our results were very similar to that of others for a similar period [Citation13,Citation14] and this was despite the fact that the Karolinska University Hospital covered roughly 60% of the SCC and ADC cases in the Stockholm area. Another limitation was that the available samples were from patients with a median age slightly below that of the median age of the patients in the Swedish Cancer Registry for the studied region. This is likely due to that there were fewer samples with sufficient material from the older patients. Nevertheless, this age difference was marginal and similar to that reported by others [Citation14,Citation23] which should not affect the general relevance of the study. Finally, using paraffin-embedded material could be considered as a limitation in this study. However, we have previously tested for HPV in historical paraffin-embedded tissue (tonsillar cancer from 1970–2006) and shown that in paraffin-embedded material less than 10 years of age virtually all samples, as also here, had PCR amplifiable DNA [Citation24,Citation25].
In summary, this study describes a high prevalence of HR HPV and of HPV16 and 18, in SCC and ADC in the Stockholm region, which is comparable to many European studies. In addition, the fact that HPV31, 33, 45 and 52, which are potentially covered by the present HPV vaccine, account for around another 11.7% of the tumours, which suggests that a large proportion of today's tumours can be prevented by HPV vaccination. This information thus underlines the urgency to initiate HPV vaccination, which so far has not been started in Sweden.
Acknowledgements
The Swedish Cancer Society, The Swedish Medical Research Council, The Stockholm Cancer Society, the Karolinska Institutet, The Swedish Institute for Infectious Disease Control, and the Stockholm City Council are greatly acknowledged for their financial support. There are no conflicts of interest. Although support has been received from The Swedish Institute for Infectious Disease Control and the Karolinska Institute, both are government institutes.
Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.
References
- Ferlay J, Shin HR, Bray F, Forman D, Mathers C, Parkin DM. Estimates of worldwide burden of cancer in 2008: GLOBOCAN 2008. Int J Cancer 2010;127:2893–917.
- Gustafsson L, Ponten J, Zack M, Adami HO. International incidence rates of invasive cervical cancer after introduction of cytological screening. Cancer Causes Control 1997;8:755–63.
- Engholm G, Ferlay J, Christensen N, Bray F, Gjerstorff ML, Klint A, . NORDCAN-a Nordic tool for cancer information, planning, quality control and research. Acta Oncol 2010;49:725–36.
- Bray F, Carstensen B, Moller H, Zappa M, Zakelj MP, Lawrence G, . Incidence trends of adenocarcinoma of the cervix in 13 European countries. Cancer Epidemiol Biomarkers Prev 2005;14:2191–9.
- Parkin DM, Whelan SL, Ferlay J, Teppo L, Thomas DB. Cancer incidence in five continents. Volume VIII. IARC Sci Publ 2002;155:1–781.
- Bosch FX, Lorincz A, Munoz N, Meijer CJ, Shah KV. The causal relation between human papillomavirus and cervical cancer. J Clin Pathol 2002;55:244–65.
- Castellsague X, de Sanjose S, Aguado T, Louie KS, Bruni L, Munoz J, . HPV and cervical cancer in the 2007 report. Vaccine 2007;25(Suppl 3):C1–230.
- Walboomers JM, Jacobs MV, Manos MM, Bosch FX, Kummer JA, Shah KV, . Human papillomavirus is a necessary cause of invasive cervical cancer worldwide. J Pathol 1999;189:12–9.
- Li N, Franceschi S, Howell-Jones R, Snijders PJ, Clifford GM. Human papillomavirus type distribution in 30,848 invasive cervical cancers worldwide: Variation by geographical region, histological type and year of publication. Int J Cancer 2011;128:927–35.
- Clifford GM, Smith JS, Plummer M, Munoz N, Franceschi S. Human papillomavirus types in invasive cervical cancer worldwide: A meta-analysis. Br J Cancer 2003;88:63–73.
- de Sanjose S, Quint WG, Alemany L, Geraets DT, Klaustermeier JE, Lloveras B, . Human papillomavirus genotype attribution in invasive cervical cancer: A retrospective cross-sectional worldwide study. Lancet Oncol 2010;11:1048–56.
- Usubutun A, Alemany L, Kucukali T, Ayhan A, Yuce K, de Sanjose S, . Human papillomavirus types in invasive cervical cancer specimens from Turkey. Int J Gynecol Pathol 2009;28:541–8.
- Carozzi FM, Tornesello ML, Burroni E, Loquercio G, Carillo G, Angeloni C, . Prevalence of human papillomavirus types in high-grade cervical intraepithelial neoplasia and cancer in Italy. Cancer Epidemiol Biomarkers Prev 2010;19:2389–400.
- Howell-Jones R, Bailey A, Beddows S, Sargent A, de Silva N, Wilson G, . Multi-site study of HPV type-specific prevalence in women with cervical cancer, intraepithelial neoplasia and normal cytology, in England. Br J Cancer 2010;103:209–16.
- Mariani L, Monfulleda N, Alemany L, Vizza E, Marandino F, Vocaturo A, . Human papillomavirus prevalence and type-specific relative contribution in invasive cervical cancer specimens from Italy. BMC Cancer 2010;10:259.
- Garland SM, Smith JS. Human papillomavirus vaccines: Current status and future prospects. Drugs 2010;70:1079–98.
- Diaz ML. Prevention of cervical, vaginal, and vulval cancers: Role of the quadrivalent human papillomavirus (6, 11, 16, 18) recombinant vaccine. Int J Womens Health 2010;1:119–29.
- Brown DR, Kjaer SK, Sigurdsson K, Iversen OE, Hernandez-Avila M, Wheeler CM, . The impact of quadrivalent human papillomavirus (HPV; types 6, 11, 16, and 18) L1 virus-like particle vaccine on infection and disease due to oncogenic nonvaccine HPV types in generally HPV-naive women aged 16–26 years. J Infect Dis 2009;199:926–35.
- Munoz N, Bosch FX, de Sanjose S, Herrero R, Castellsague X, Shah KV, . Epidemiologic classification of human papillomavirus types associated with cervical cancer. N Engl J Med 2003;348:518–27.
- Schmitt M, Bravo IG, Snijders PJ, Gissmann L, Pawlita M, Waterboer T. Bead-based multiplex genotyping of human papillomaviruses. J Clin Microbiol 2006;44:504–12.
- Ramqvist T, Du J, Lunden M, Ahrlund-Richter S, Ferreira J, Marions L, . Pre-vaccination prevalence of human papillomavirus types in the genital tract of 15–23-year-old women attending a youth health clinic in Stockholm, Sweden. Scand J Infect Dis 2011;43:115–21.
- Dahlstrom LA, Ylitalo N, Sundstrom K, Palmgren J, Ploner A, Eloranta S, . Prospective study of human papillomavirus and risk of cervical adenocarcinoma. Int J Cancer 2010;127:1923–30.
- Andersson S, Rylander E, Larsson B, Strand A, Silfversvard C, Wilander E. The role of human papillomavirus in cervical adenocarcinoma carcinogenesis. Eur J Cancer 2001;37:246–50.
- Hammarstedt L, Lindquist D, Dahlstrand H, Romanitan M, Dahlgren LO, Joneberg J, . Human papillomavirus as a risk factor for the increase in incidence of tonsillar cancer. Int J Cancer 2006;119:2620–3.
- Nasman A, Attner P, Hammarstedt L, Du J, Eriksson M, Giraud G, . Incidence of human papillomavirus (HPV) positive tonsillar carcinoma in Stockholm, Sweden: An epidemic of viral-induced carcinoma? Int J Cancer 2009;125:362–6.