University of Groningen. New insights in methodology of screening for cervical cancer Wang, Rong

University of Groningen New insights in methodology of screening for cervical cancer Wang, Rong IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite from it. Please check the document version below. Document Version Publisher's PDF, also known as Version of record Publication date: 2015 Link to publication in University of Groningen/UMCG research database Citation for published version (APA): Wang, R. (2015). New insights in methodology of screening for cervical cancer: Nieuwe inzichten in de methodologie van baarmoederhalskanker screening [Groningen]: University of Groningen Copyright Other than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons). Take-down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons the number of authors shown on this cover page is limited to 10 maximum. Download date: 31-12-2017

Summary and future perspectives Chapter 6 Summary and future perspectives 141

Chapter 6 Summary Although cytomorphology-based cervical screening has reduced the incidence of cervical cancer in the Western world, it is predicted that by 2030 the number of deaths from cervical cancer will increase to 410,000 worldwide 1,2,3. The progress in prevention, early detection and treatment of cervical cancer has been hampered by the fact that the majority of cases occur in the developing world where infrastructure, human resources and medical facilities with sufficient quality assurance are limited or absent 4. As one of the developing countries, China plays an important role in the global fight against cervical cancer. However, several challenges play a major role in this fight such as the huge population and area, the rapid, unbalanced economic growth and the mass migration of the last years. Due to its low sensitivity and high labor-intensity, cytomorphology-based examination of cervical smears may not be an ideal screening method in China 5. HPV testing is another well-known approach for early detection of cervical cancer. Introduction in China will generate a huge and chaotic market for numerous HPV detection platforms with different specifications and therefore different (dis)advantages. It will be necessary to choose an appropriate and validated platform before HPV detection is introduced as a primary screening tool. Simultaneously, another important question to be answered first is how to manage the HPV positive patient, particular for the densely populated, but less-developed regions. Therefore, to explore feasible cervical screening tools which may cover and benefit the large cohorts effectively, new insights in cervical cancer biomarkers including HPV testing and DNA methylation markers are investigated and discussed in this thesis. Globally HPV DNA screening has been performed for more than 15 years and demonstrated that the prevalence of HPV infection varies geographically and racially 6,7. In China, although several projects regarding the hrhpv infection rate and cervical cancer screening were conducted 8 (Table 4 in Chapter 1), the data are still far from a nationwide standard and this information should be updated with time in order to provide reference for effective screening. In Chapter 2 we described a nationwide cross-sectional investigation to investigate HPV prevalence in most regions in China, to follow up the potential changes of HPV infection in the regions 142

Summary and future perspectives previously studied and to clarify the genotypic distribution of HPV in different regions and age-grouped populations. In this study 120,772 liquid-based cytological samples from women enrolled for population- or employee-based cervical screening programs in 37 Chinese cities in 2012, were obtained and sent to the laboratory of molecular infectious diseases of Guangzhou KingMed. Overall 111,131 samples were tested by Hybrid Capture 2 and 9,641 samples were genotyped by Tellgenplex TM HPV DNA Assay. The total positive rate of high-risk HPV (hrhpv) was 21.07%, ranging from 31.94% to 18.42% in different regions. Age-specific prevalence showed a two peak pattern. The youngest age-group (15-19 years) presented the highest hrhpv infection rate (30.55%) and the second peak was found in the age group of 50-60 years. HPV16 (4.82% of all samples) and HPV52 (4.52% of all samples) were the most prevalent hrhpv types, followed by HPV58 (2.74% of all samples); while HPV6 (4.01% of all samples) and HPV11 (2.29% of all samples) were predominant in the low risk HPV (lrhpv) types. HPV16+HPV52 and HPV52+HPV58 were the most common coexistence genotypes in case of multiple infections. In conclusion, HPV infection rate in China has increased into the levels of HPV-heavy-burden country zones and the rates have changed depending on regions along with time. Therefore, this investigation provides an updated and valuable reference for an effective nationwide cervical cancer screening program. Particularly, the analysis of genotypic distribution of hrhpv has again demonstrated that HPV52 and HPV58 are two of the dominant genotypes in China, which underlines that the currently available commercial vaccines, Cervarix (HPV16/18) and Gardasil (HPV6/11/16/18) may not be fully effective within the Chinese population. These epidemiological characteristics should be taken into account when considering implementation of screening and vaccination programs in China. Currently, many new screening technologies are developed and marketed in China with no validation of their effectiveness for population-based screening including HPV assays 9. A few investigators are trying to properly evaluate algorithms that can be applied to the highest-risk areas in China as well as the more urban areas. Today many commercial HPV DNA detection assays are available but only few have been formally validated in primary cervical screening including the Hybrid Capture 2 (HC2) hrhpv DNA test, the first hrhpv test approved by FDA in the US 11. The Cervista 143

Chapter 6 HR HPV test (Hologic Inc), the second hrhpv assay approved by the FDA is widely used in the US and in some Chinese studies. Cervista has a few advantages over HC2: It requires less sample volume, includes an internal control, and has a shorter processing time. However, Cervista has not previously been formally validated in primary cervical screening. In Chapter 3, we demonstrated that Cervista fulfilled the cross-sectional clinical performance and reproducibility criteria of international guidelines for HPV test requirements for cervical screening. By non-inferiority analysis following international guidelines the clinical sensitivity and specificity of Cervista were compared to that of HC2 for detection of high-grade cervical lesions (CIN2+) in Chinese women aged 30 years in >7,000 cervical screening samples selected from a multisite, population-based, cross-sectional study. Cervista showed a clinical sensitivity for CIN2+ of 89% (95%CI: 81.7-93.6) and a clinical specificity for CIN2+ of 91% (95%CI: 90.5-91.8). Both the relative clinical sensitivity and specificity were non-inferior to that of HC2 (non-inferiority score tests, p=0.043 and p<0.0001, respectively). In addition, we also determined the intra- and inter-laboratory reproducibility of Cervista in 510 scrapings, again following international guidelines. Intra- and inter-laboratory agreements were 92% (lower bound 95%CI: 89.7%; kappa = 0.83; p<0.001) and 90.4% (lower bound 95%CI: 88.4%; kappa = 0.81; p<0.001), respectively. In conclusion, the Cervista HR HPV test met the cross-sectional clinical and reproducibility criteria of the international guidelines for HPV test requirements and can be considered as clinically validated for primary cervical screening purposes. Screening identifies not only early stage invasive cancers that are expected to be more amenable to cure, but also pre-malignancies that easily can be removed and thus prevented from progressing to invasive lesions 10. However, the relatively lower specificity of hrhpv testing, especially in a young screening population, may lead to unnecessary referrals to the gynecologist, anxiety in the false-positive women, and higher costs for the health-care system and indeed is a challenge for cervical screening programs. In addition, in the near future the prevalence of CIN and cervical cancer will probably decrease in countries, that have introduced primary prevention with hrhpv vaccination. With this decrease in prevalence, the positive predictive value of the current screening tests will by definition decrease. 144

Summary and future perspectives Therefore, other objective biomarkers with both high sensitivity as well as high specificity are needed as new screening or triage tools for cervical cancer. Differential DNA methylation patterns in normal cervical epithelium versus (pre)malignant cervical lesions represent excellent targets for diagnostic approaches based on methylation specific PCR (MSP). In our previous studies, the pharmacological unmasking of the promoter region combined with re-expression as analyzed by microarrays, QMSP on an OpenArray platform and methyl-dna immunoprecipitation followed by microarray analysis (MeDIP), resulted in the discovery of 4 genes (C13ORF18, JAM3, EPB41L3 and TERT) 11,12,13. The diagnostic performance of DNA methylation analysis of these genes showed sensitivities for detecting CIN2+ in a hrhpv positive population between 43-71% and specificities between 89-100% 11. However, our strategies for discovering new methylation markers so far were based on the difference between cancer and normal tissue, resulting in markers with high sensitivity for carcinomas, but with too low sensitivity for detecting CIN2/3 lesions. In Chapter 4, in order to identify new methylation markers for high-grade cervical intraepithelial neoplasia (CIN2/3), a novel technique (MethylCap-seq) was applied to identify differential methylation regions (DMRs) and generate a CIN2/3 lesion-specific methylome. Comparing 20 normal cervices with 18 CIN2/3 lesions, 176 DMRs comprising 163 genes were identified. The top 15 highest ranking differentially methylated genes were selected and validated by methylation specific PCR (MSP) in two steps: on the same DNA samples as used for MethylCapseq and on DNA samples from an independent patient cohort with (pre)malignant cervical neoplasia. For further diagnostic evaluation, the best differentiating methylation markers were tested with quantitative MSP (QMSP) on cervical scrapings from 2 cohorts: 1) cervical carcinomas versus healthy controls and 2) patients referred from population-based screening with an abnormal Pap smear in whom HPV status was determined. After verification and validation of the top 15 genes with MSP, 9 genes showed significant differential methylation in normal cervices versus CIN2/3 lesions (p<0.05). Subsequently, methylation levels of 8/9 genes were significantly higher in carcinomas compared to normal scrapings. For all 8 genes methylation levels increased with the 145

Chapter 6 severity of the underlying histological lesion in scrapings from patients with an abnormal Pap smear. In addition to the 8 new genes, also methylation levels of our previous four-gene panel (C13ORF18, JAM3, EPB41L3 and TERT) were analyzed on the same samples. The combination C13ORF18/JAM3/AL590705.4 revealed a similar high sensitivity for CIN2+ (74%) compared to hrhpv testing (79%), while specificity was significantly higher (76%) compared to hrhpv testing (42%) (p 0.05). With this genome-wide DNA methylation analysis we identified new CIN2/3 specific methylation markers. The diagnostic performance of our new methylation panel showed comparable sensitivity to hrhpv testing for CIN2+, but with higher specificity, therefore possibly preventing unnecessary referral for colposcopy. The next step before implementation in primary screening programs will be validation of our new methylation panel in population-based cohorts. Cervical cancer encompasses several histological types, of which adenocarcinomas (ADC) and cervical squamous cell carcinoma (SCC) are the two main subtypes, accounting for 10 25% and 75 90%, respectively 14,15,16. Currently, the incidence of SCC is declining in most developed countries, while there is a rise in the absolute and relative incidence of ADC 15,17. Although ADC is mainly increasing rapidly in Europe, and relatively slower in Asia (2.8-22.6%) 16, the absolute cases of ADC still cannot be ignored, because of its large population. Moreover, in comparison with SCC, ADC is mainly diagnosed at a more advanced stage of disease 16,17. Current screening programs are more effective in detection of the precursors of SCC than those of ADC. Major reason for the inefficacy of current screening methodology for detection of ADC is its location higher up in the cervical canal, which hampers acquisition of adequate samples for cytology and also direct observation by colposcopic examination 17. Next to hrhpv testing, analysis of DNA methylation markers might improve the detection of ADC in earlier stages. However, no specific methylation markers have been described for the detection of ADC as well as for SCC. The aim of the study described in Chapter 5 was to discover novel methylation biomarkers for cervical cancer detecting both ADC and SCC. Novel methylation markers were identified using MethylCap-seq by comparison of 20 normal cervices with 12 cancers (6 ADC, 6 SCC). The top 15 markers were selected for verification by bisulfite pyrosequencing or MSP on the same samples used for MethylCap-seq. 146

Summary and future perspectives Methylation frequencies were validated on a series of paraffin-embedded specimens from normal cervices and cervical cancers by MSP. Finally, QMSP was performed on cervical scrapings from an independent cohort of women with a normal cervix and cervical cancer. Validation of the highest ranking 15 differentially methylated candidate genes resulted in 5 markers exhibiting different methylation frequencies between normal and cancer tissues (p<0.05). Using QMSP on DNA of cervical scrapings, the sensitivity of these 5 markers varied from 80.5% to 91.9% to detect both ADC and SCC with almost all normal scrapings negative (specificity: 94% - 98.9%). In conclusion, using the MethylCap-seq analysis, we identified 5 new methylation markers for early detection of both ADC and SCC in cervical scrapings. Further validation in a large series of population-based scrapings is needed, in particular in scrapings from ADC and its precursors, to confirm our findings. Future perspectives A successful and effective national cervical cancer screening program depends on an optimal coverage of the screening population, high-level quality assurance of the screening methodology and proper management of positive findings 18,19. Recently, many observational studies 20,21,22 and a systematic meta- analysis 23 have demonstrated that a cervical screening program effectively will reduce the morbidity and mortality of cervical cancer in most high-income settings. However, cytologybased screening has reached a plateau owing to the low sensitivity, low reproducibility, highly variable results among laboratories and poor performance in detection of cervical adenocarcinoma 24,25,26. Liquid-based-cytology (LBC) is considered to be superior over conventional Pap cytology by its standardization, resulting in easier handling and higher reproducibility with a similar sensitivity for the detection of CIN2/3 and adenocarcinomas 27. The strong link between HPV and cervical carcinogenesis paves new ways for cervical cancer prevention. Immunization for hrhpv as a means of primary prevention and HPV DNA testing in cervical screening for the early detection of (pre)malignant lesions are now widely applied. In 2006, Gardasil, a quadrivalent 147

Chapter 6 vaccine and Cervarix, a bivalent vaccine were fully licensed 28. Until the beginning of 2012, around 40 countries have introduced HPV vaccination into their national immunization program 28. However, there are still some obstacles with respect to the currently approved vaccines that limit their efficiency: 1) Immunization may fail to protect a significant proportion of women, because with the current vaccines women will only be effectively protected against HPV16 and HPV18. On the other hand, many other hrhpv genotypes have been detected and the prevalence of the different other hrhpv genotypes varies in different countries and regions within countries 6. For example, we found (Chapter 2) that HPV52 and HPV58 have the highest prevalence after HPV16 in most of the regions in China. Therefore, using the more recently developed polyvalent HPV vaccine against HPV6, HPV11, HPV16, HPV18, HPV31, HPV33, HPV45, HPV52 and HPV58 (Merck s Investigational 9-valent HPV Vaccine V503) might be more effective in immunization programs in China 29 30. 2) The costs of the approved HPV vaccines has also been a hurdle in the introduction of the vaccines, especially in developing countries which carry the greatest burden of HPVassociated diseases 31. 3) Concerns about implications for young women s future sexual, physical and reproductive health, parental refusal or religious beliefs are all factors that influence participation rates in the vaccination program 32. Despite HPV vaccination, cervical screening will remain an important aspect of cervical cancer prevention, both for the vaccinated and non-vaccinated women. In the Netherlands, the prophylactic vaccine Cervarix against HPV16 and 18 was introduced in the national immunization program in 2009 33. However, as the current vaccines are being given to adolescent girls only, it will take 10-15 years before the effect of vaccination on cervical cancer prevalence becomes clear. Furthermore, not all adolescent girls are vaccinated, since only 50-60% of the girls show up for the vaccination program in the Netherlands. Moreover, even vaccinated girls should be screened when they reach the screening age since the current vaccine will not provide complete protection against all oncogenic HPV types 34. Therefore, continuation of cervical cancer screening will remain necessary. 148

Summary and future perspectives Apart from the vaccines against HPV16 and 18, hrhpv DNA is an important molecular biomarker for the detection of cervical cancer and its precursors. Compared to cytology-based screening methods, clinical trials have shown that HPVbased screening results in greater sensitivity (range 94.1 95.4%) than cytologybased screening (range 55.4 71.3%) with some loss in specificity for CIN2+ (CIN2+; cytology specificity range 96.8 98.6% compared with HPV 94.1 94.2%) 35,36,37,38. However, HPV-based testing has a far better negative predictive ability than cytology-based testing. A negative HPV test immediately gives a reassurance close to 100% for absence of disease (cytology detection less than 60%) and almost guarantees protection for the absence of HSIL over a prolonged period, safely allowing lengthening of the screening interval for at least 5 years 39,40. So, the results from a HPV test provide significantly more valuable information from a disease screening perspective. In addition, compared to cytomorphology screening, molecular markers are convenient, cheap, user-friendly and less labor intensive, especially when using high-throughput automated platforms. These advantages make them also feasible for the low-resource settings, avoiding logistical complexity and expertise required by cytology. In China prophylactic vaccines have not officially been approved yet. Therefore, at this moment the most feasible strategy for cervical cancer prevention and control is organization of a national population-based screening program, thereby promoting diagnosis and treatment of cervical neoplasia at an earlier stage 41. Since nearly 70% of Chinese rural women live in poor regions lacking financial resources and more than 80% women diagnosed with cervical cancer have never been screened, the Chinese government has executed a pilot program for free cervical cancer screening for rural women between 2009-2011 42. This program has been continued for another 3 years (2012-2015), because only 10 out of 500 million Chinese rural women could access this service. Meanwhile, in accordance with the feasibility and costeffectiveness evaluation, the Ministry of Health of China has launched a guideline for region-driven screening protocols by using the cytology test plus HPV test in the more developed regions and visual inspection with acetic acid (VIA) in low-resource settings 43. As China is a large population country, cytology-based technique is not an ideal screening method because of the requirement of a large number of experienced 149

Chapter 6 cytologists/pathologists and maintaining the large numbers of clinics required for mass population screening. As to VIA, a screen-and-treat method was recommended for a large number of high-risk cases from patients living in lowresource regions. However, due to the low sensitivity of VIA, cancer patients may be missed, while on the other hand, because of no quality control, there is also a risk for overtreatment of the women due to false positive results 44. Therefore, it would be more effective to introduce objective, reproducible and high-throughput molecular biomarkers assays instead of VIA and/or morphology testing as the screening method in China. In the Netherlands, one of the leading countries in cervical cancer prevention, primary hrhpv-based screening will start in 2016 45. However, because of the much higher sensitivity of HPV testing, but relatively lower specificity, more patients with false-positive results will be referred to gynecologists, which may result in unnecessary higher costs and more unwanted anxiety of involved women. So, the question arises how to manage women who test hrhpv positive. Analysis of DNA methylation markers is objective, can be implemented in high-throughput systems and analyzed on the same DNA sample as used for the HPV assay, which presents DNA methylation analysis as an attractive triage testing method for HPV-positive patients. So far, several DNA methylation markers have been successfully identified for cervical neoplasia by our group 11,12,46 including the markers as described in this thesis. However, before our markers will be ready for clinical practice, a gradual validation process should be performed. Pepe et al. 47 recommended a five-phase framework: After preclinical exploratory studies, assessment in non-invasive samples, retrospective longitudinal studies, prospective screening studies and prospective intervention studies should be performed. Our methylation markers (Chapter 4) to detect CIN2/3 lesions were already tested in samples obtained from the present cytology-based cervical screening program. Therefore, as a next step for future HPV DNA based screening programs further validation of our markers needs to be performed in hrhpv positive scrapings for which histological data is also available, collected from a prospective population-based screening program. In the Netherlands this type of scrapings will become available after 2016 when primary cervical screening has changed to hrhpv testing. Likewise, our marker panel (Chapter 5) for 150

Summary and future perspectives the early detection of both ADC and SCC should be validated in a screening setting, particular for the precursor stage of ADC (AdCIS). In China, so far there is one population-based study 48 that reported the methylation frequency of 3 genes (DAPK1, RAR- 2 and MGMT) using liquid-based cytology samples from a large cohort. Another study identified several hypermethylated genes using captured methylated DNA combined with CpG-microarray analysis 49. Most other Chinese studies 50 51 52 53 54 on methylation markers are based on cell lines and tissue samples and therefore cannot be translated to cervical cancer screening directly. Because the distribution of HPV genotypes differs between the Chinese population (Chapter 2) and Western world, it might well be that the specificity and sensitivity for detection of CIN2+ lesions using our methylation panel will be different in a Dutch and Chinese population. Therefore, clinical validation on Chinese HPVpositive scrapings is required. Last but not least, transient HPV infections are common, especially among younger women 55. In chapter 2 we reported that the youngest age-group (15-19 years) presented the highest hrhpv infection rate (30.55%) in a Chinese screening cohort. As a result, in young women hrhpv testing to discriminate between HPV-positive scrapings with CIN2+ lesions and HPV-positive scrapings of those women with transient HPV infections only is very challenging. For that reason, some international guidelines stated that hrhpv DNA testing for screening purposes should not be performed below the age of 30 years 56,57,58. Therefore, in the Netherlands the cervical screening program starts at the age of 30 years but in many other countries (e.g. in UK) screening programs start at the age of 25. According to the current Chinese cervical cancer screening guideline, the screening program in China even starts at the age of 21 years. Hence, a DNA methylation biomarker panel could be very relevant to identify women aged 21-29 years with HPV-positive scrapings and with CIN2+ lesions, thereby excluding those women with transient HPV infections that are not associated with CIN2+ lesions. The validation of the relevance of methylation markers to identify Chinese women younger than 30 years with CIN2+ lesions therefore needs to be performed in future studies. 151

Chapter 6 Reference 1 Kamangar F, Dores GM, Anderson WF. Patterns of cancer incidence, mortality, and prevalence across five continents: defining priorities to reduce cancer disparities in different geographic regions of the world. J Clin Oncol 2006; 24: 2137 50. 2 Mathers C, Boerma T MFD. The global burden of disease: 2004 update (Geneva, Switzerland: World Health Organization). http://www.who.int/healthinfo/global_burden_disease/gbd_report_2004update_full.pdf. 3 Thomas G. Are we making progress in curing advanced cervical cancer? J Clin Oncol 2011; 29: 1654 6. 4 Nogueira-Rodrigues A, Ferreira CG, Bergmann A, de Aguiar SS, Thuler LCS. Comparison of adenocarcinoma (ACA) and squamous cell carcinoma (SCC) of the uterine cervix in a suboptimally screened cohort: A population-based epidemiologic study of 51,842 women in Brazil. Gynecol Oncol 2014; published online Aug 14. DOI:10.1016/j.ygyno.2014.08.014. 5 Wright TC, Kuhn L. Alternative approaches to cervical cancer screening for developing countries. Best Pract Res Clin Obstet Gynaecol 2012; 26: 197 208. 6 De Sanjosé S, Diaz M, Castellsagué X, 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: 453 9. 7 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: 1789 99. 8 Li J, Huang R, Schmidt JE, Qiao YL. Epidemiological Features of Human Papillomavirus (HPV) Infection among Women Living in Mainland China. Asian Pac J Cancer Prev 2013; 14: 4015 23. 9 Castle PE, de Sanjose S, Qiao Y-LL, et al. Introduction of human papillomavirus DNA screening in the world: 15 years of experience. Vaccine 2012; 30 Suppl 5: F117 22. 10 Umar A, Dunn BK, Greenwald P. Future directions in cancer prevention. Nat Rev Cancer 2012; 12: 835 48. 11 Eijsink JJH, Lendvai Á, Deregowski V, et al. A four-gene methylation marker panel as triage test in high- risk human papillomavirus positive patients. Int J cancer 2012; 1869: 1861 9. 12 Yang N, Eijsink JJH, Lendvai A, et al. Methylation markers for CCNA1 and C13ORF18 are strongly associated with high-grade cervical intraepithelial neoplasia and cervical cancer in cervical scrapings. Cancer Epidemiol Biomarkers Prev 2009; 18: 3000 7. 13 Lendvai Á, Johannes F, Grimm C, et al. Genome-wide methylation profiling identifies hypermethylated biomarkers in high-grade cervical intraepithelial neoplasia. Epigenetics 2012; 7: 1268 78. 152

Summary and future perspectives 14 Vizcaino AP, Moreno V, Bosch FX, et al. International trends in incidence of cervical cancer: II. Squamous-cell carcinoma. Int J cancer 2000; 86: 429 35. 15 Vizcaino AP, Moreno V, Bosch FX, Muñoz N, Barros-Dios XM, Parkin DM. International trends in the incidence of cervical cancer: I. Adenocarcinoma and adenosquamous cell carcinomas. Int J cancer 1998; 75: 536 45. 16 Seoud M, Tjalma W, Ronsse V. Cervical adenocarcinoma: moving towards better prevention. Vaccine 2011; 29: 9148 58. 17 Gien L, Beauchemin M, Thomas G. Adenocarcinoma: a unique cervical cancer. Gynecol Oncol 2010; 116: 140 6. 18 Attila. New strategies for HPV-based cervical screening. Womens Heal (l Engl) 2014; 9: 1 16. 19 Van Bogaert L. Are the currently existing anti-human papillomavirus vaccines appropriate for the developing world? Ann Med Health Sci Res 2013; 3: 306 12. 20 Quinn M, Babb P, Jones J, Allen E. Effect of screening on incidence of and mortality from cancer of cervix in England: evaluation based on routinely collected statistics. BMJ 1999; 318: 904 8. 21 Peto J, Gilham C, Fletcher O, Matthews FE. The cervical cancer epidemic that screening has prevented in the UK. Lancet; 364: 249 56. 22 Smith-Bindman R, Chu PW, Miglioretti DL, et al. Comparison of screening mammography in the United States and the United kingdom. JAMA 2003; 290: 2129 37. 23 Peirson L, Fitzpatrick-Lewis D, Ciliska D, Warren R. Screening for cervical cancer: a systematic review and meta-analysis. Syst Rev 2013; 2: 35. 24 ACOG Practice Bulletin no. 109: Cervical cytology screening. Obstet Gynecol 2009; 114: 1409 20. 25 Herzog TJ, Monk BJ. Reducing the burden of glandular carcinomas of the uterine cervix. Am J Obstet Gynecol 2007; 197: 566 71. 26 Baker E. HPV and Pap: shifting roles in cervical cancer screening. Med Lab Obs 2013; 45: 24, 26. 27 Arbyn M, Bergeron C, Klinkhamer P, Martin-Hirsch P, Siebers AG, Bulten J. Liquid compared with conventional cervical cytology: a systematic review and meta-analysis. Obstet Gynecol 2008; 111: 167 77. 28 Markowitz LE, Tsu V, Deeks SL, et al. Human papillomavirus vaccine introduction--the first five years. Vaccine 2012; 30 Suppl 5: F139 48. 29 Serrano B, Alemany L, Alonso P, et al. Potential impact of a 9-valent HPV vaccine in HPVrelated cervical disease in 4 emerging countries ( Brazil, Mexico, India and China ). Cancer Epidemiol 2014; 38: 748 56. 153

Chapter 6 30 Joura EA, Ault KA, Bosch FX, Brown D, Cuzick J, Ferris D, Garland SM, Giuliano AR, Hernandez-Avila M, Huh W, Iversen OE, Kjaer SK, Luna J, Miller D, Monsonego J, Munoz N, Myers E, Paavonen J, Pitisuttithum P, Steben M, Wheeler CM, Perez G, Saah A, Luxembo VC. Attribution of 12 high-risk human papillomavirus genotypes to infection and cervical disease. Cancer Epidemiol Biomarkers Prev 2014; 23: 1997 2008. 31 Financing HPV vaccination in developing countries. Lancet 2011; 377: 1544. 32 Ferrer HB, Trotter C, Hickman M, Audrey S. Barriers and facilitators to HPV vaccination of young women in high-income countries: a qualitative systematic review and evidence synthesis. BMC Public Health 2014; 14: 700. 33 Westra TA, Rozenbaum MH, Rogoza RM, et al. Until which age should women be vaccinated against HPV infection? Recommendation based on cost-effectiveness analyses. J Infect Dis 2011; 204: 377 84. 34 Harper DM, Franco EL, Wheeler CM, et al. Sustained efficacy up to 4.5 years of a bivalent L1 virus-like particle vaccine against human papillomavirus types 16 and 18: follow-up from a randomised control trial. Lancet 2006; 367: 1247 55. 35 Naucler P, Ryd W, Törnberg S, et al. Efficacy of HPV DNA testing with cytology triage and/or repeat HPV DNA testing in primary cervical cancer screening. J Natl Cancer Inst 2009; 101: 88 99. 36 Mayrand M-H, Duarte-Franco E, Rodrigues I, et al. Human papillomavirus DNA versus Papanicolaou screening tests for cervical cancer. N Engl J Med 2007; 357: 1579 88. 37 Ronco G, Giorgi-Rossi P, Carozzi F, et al. Results at recruitment from a randomized controlled trial comparing human papillomavirus testing alone with conventional cytology as the primary cervical cancer screening test. J Natl Cancer Inst 2008; 100: 492 501. 38 Elfström KM, Herweijer E, Sundström K, Arnheim-Dahlström L. Current cervical cancer prevention strategies including cervical screening and prophylactic human papillomavirus vaccination: a review. Curr Opin Oncol 2014; 26: 120 9. 39 Monsonego J. HPV testing in prevention of cervical cancer: practices and current trends. Ann Biol Clin (Paris) 2013; 71: 27 32. 40 Giorgi Rossi P, Baldacchini F, Ronco G. The Possible Effects on Socio-Economic Inequalities of Introducing HPV Testing as Primary Test in Cervical Cancer Screening Programs. Front Oncol 2014; 4: 20. 41 Liu XY, Feng AH, Cui YM, Tobe RG. Prevention of human papillomavirus (HPV) infection and cervical cancer in China: How does HPV vaccination bring about benefits to Chinese women? Biosci Trends 2013; 7: 159 67. 42 Women s health in rural China. Lancet 2009; 374: 358. 154

Summary and future perspectives 43 Zhao FH, Lewkowitz AK, Hu SY, et al. Prevalence of human papillomavirus and cervical intraepithelial neoplasia in China: a pooled analysis of 17 population-based studies. Int J Cancer 2012; 131: 2929 38. 44 Cervical Cancer Screening in Developing Countries: report of a WHO consultation. http://www.who.int/cancer/media/en/cancer_cervical_37321.pdf. 45 Boers A, Slagter-Menkema L, van Hemel BM, et al. Comparing the Cervista HPV HR Test and Hybrid Capture 2 Assay in a Dutch Screening Population: Improved Specificity of the Cervista HPV HR Test by Changing the Cut-Off. PLoS One 2014; 9: e101930. 46 Boers A, Bosgraaf RP, van Leeuwen RW, et al. DNA methylation analysis in self-sampled brush material as a triage test in hrhpv-positive women. Br J Cancer 2014; 111: 1095 101. 47 Pepe MS, Etzioni R, Feng Z, et al. Phases of biomarker development for early detection of cancer. J Natl Cancer Inst 2001; 93: 1054 61. 48 Sun LL, Cao DY, Yang JX, et al. Population-based case-control study on DAPK1, RAR-beta2 and MGMT methylation in liquid-based cytology. Arch Gynecol Obs 2012; 285: 1433 9. 49 Wu JH, Liang XA, Wu YM, Li FS, Dai YM. Identification of DNA methylation of SOX9 in cervical cancer using methylated-cpg island recovery assay. Oncol Rep 2013; 29: 125 32. 50 Wu Y, Meng L, Wang H, et al. Regulation of DNA methylation on the expression of the FHIT gene contributes to cervical carcinoma cell tumorigenesis. Oncol Rep 2006; 16: 625 9. 51 Song Y, Zhang C. Hydralazine inhibits human cervical cancer cell growth in vitro in association with APC demethylation and re-expression. Cancer Chemother Pharmacol 2009; 63: 605 13. 52 WANG, SS., WANG N., YU X., YANG CX., YAN LP. WY. Study on methylation status of RASSFIA gene promoter and exon 1 in cervical cancer cell lines. China Oncol 2013; 23: 777 83. 53 Chen CL, Liu SS, Ip SM, Wong LC, Ng TY, Ngan HY. E-cadherin expression is silenced by DNA methylation in cervical cancer cell lines and tumours. Eur J Cancer 2003; 39: 517 23. 54 Yu MY, Tong JH, Chan PK, et al. Hypermethylation of the tumor suppressor gene RASSFIA and frequent concomitant loss of heterozygosity at 3p21 in cervical cancers. Int J Cancer 2003; 105: 204 9. 55 Kitchener HC, Almonte M, Thomson C, et al. HPV testing in combination with liquid-based cytology in primary cervical screening (ARTISTIC): a randomised controlled trial. Lancet Oncol 2009; 10: 672 82. 56 Arbyn M, Anttila A, Jordan J, et al. European Guidelines for Quality Assurance in Cervical Cancer Screening. Second edition--summary document. Ann Oncol 2010; 21: 448 58. 57 Wright TC, Massad LS, Dunton CJ, Spitzer M, Wilkinson EJ, Solomon D. 2006 consensus guidelines for the management of women with abnormal cervical cancer screening tests. Am J Obstet Gynecol 2007; 197: 346 55. 155

Chapter 6 58 Kitchener H, Denton K, Soldan K, Crosbie E. Developing role of HPV in cervical cancer prevention. BMJ 2013; 4781: 1 7. 156