BMJ Open. Primary cervical cancer screening with an HPV mrna test

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1 Primary cervical cancer screening with an HPV mrna test Journal: BMJ Open Manuscript ID bmjopen--0 Article Type: Research Date Submitted by the Author: -Mar- Complete List of Authors: Sørbye, Sveinung Wergeland; University Hospital of North Norway, Clinical Pathology Fismen, Silje; University Hospital of North Norway, Clinical Pathology Gutteberg, Tore; University Hospital of North Norway, Department of Microbiology and Infection Control; University of Tromsø, Department of Medical Biology, Faculty of Health Sciences Mortensen, Elin; University Hospital of North Norway, Clinical Pathology; University of Tromsø, Department of Medical Biology, Faculty of Health Sciences Skjeldestad, Finn Egil; Women's Health and Perinatology Research Group, University of Tromsø, Norway, Department of Clinical Medicine <b>primary Subject Heading</b>: Pathology Secondary Subject Heading: Epidemiology, Obstetrics and gynaecology, Public health, Sexual health Keywords: hpv mrna test, cervical intraepithelial neoplasia, CIN+, cervical cancer screening, hpv primary screening, hpv BMJ Open: first published as 0./bmjopen--0 on August. Downloaded from on January by guest. Protected by copyright.

2 Page of BMJ Open 0 Primary cervical cancer screening with an HPV mrna test Sveinung Wergeland Sørbye, Silje Fismen, Tore Jarl Gutteberg,, Elin Synnøve Mortensen,, Finn Egil Skjeldestad Department of Clinical Pathology, University Hospital of North Norway, Tromsø, Norway Department of Microbiology and Infection Control, University Hospital of North Norway, Tromsø, Norway Department of Medical Biology, Faculty of Health Sciences, University of Tromsø, Tromsø, Norway Institute of Community Medicine, Research Group Epidemiology of Chronic Diseases, UiT The Arctic University of Norway, Tromsø, Norway *Corresponding author and reprints, Sveinung Wergeland Sørbye, MD Department of Clinical Pathology University Hospital of North Norway 0 Tromsø, Norway Telephone, + or + 0 Fax, + 0 , sveinung.sorbye@unn.no - BMJ Open: first published as 0./bmjopen--0 on August. Downloaded from on January by guest. Protected by copyright.

3 Page of 0 Abstract Objectives: Assess the performance of -type HPV mrna test in primary screening within the framework of the Norwegian population-based screening program. Design: Nationwide register-based cohort study. Setting: In 0 0, general practitioners and gynecologists recruited women for participation in a primary screening study with a -type HPV mrna test. Participants: After excluding women with a history of abnormal smears and with CIN+ before or until months after screening, women aged years were eligible for study participation. The Norwegian Cancer Registry completed follow-up of CIN+ through December 0. Interventions: Follow-up according to the algorithm for cytology outcomes in the population-based Norwegian Cervical Cancer Screening Programme Main outcome measures: We estimated cumulative incidence of cervical intraepithelial neoplasia grade or worse (CIN+) months after -type HPV mrna test. Results:.% of the women were HPV mrna-positive at baseline. The overall cumulative rate of CIN+ was.% (% CI:..) through months of follow-up,.% for women years (n= ) and 0.% for women years (n= ). Cumulative CIN+ rates by baseline status for HPV mrna-positive and mrna-negative women years were.% (% CI:..) and 0.% (% CI: 0..), respectively, and.% (% CI: 0..) and 0.% (% CI: 0. 0.), respectively, in women years. Conclusions: The present cumulative incidence of CIN+ is similar to rates reported in screening studies via HPV DNA tests. Due to differences in biologic rationale and test characteristics, there is a trade-off between sensitivity and specificity that must be balanced when decisions on HPV tests in primary screening are taken. HPV mrna testing may be used as primary screening both for women years and years. - BMJ Open: first published as 0./bmjopen--0 on August. Downloaded from on January by guest. Protected by copyright.

4 Page of BMJ Open 0 Strengths and limitations of this study We consider studying primary screening with a -type HPV mrna test in a population of women with no previous CIN+ and/or abnormal smears as a strength as the HPV infections diagnosed are likely to be new infections. We consider the follow-up within the Norwegian Cervical Cancer Screening Programme as strength, as women regardless of mobility within Norway, are captures by the surveillance system for both cytology, histology and treatment. We consider just having one screening round with the -type HPV mrna test as a limitation, in addition to follow-up based on cytology only (verification bias). - BMJ Open: first published as 0./bmjopen--0 on August. Downloaded from on January by guest. Protected by copyright.

5 Page of 0 Introduction Cervical cancer is the third most common cancer among women worldwide. Cervical human papillomavirus (HPV) infection is necessary but not sufficient to cause cervical cancer ;. HPV is one of the most common sexually transmitted infections with a prevalence ranging from to % in young female populations ;. The lifetime risk of HPV infection in women is 0 0%. Approximately HPV genotypes, referred to as high-risk (HR) types, are considered etiological agents of cervical cancer. HPV- is by far the most significant HPV type in persisting infection and promoting progression into high-grade cervical intraepithelial neoplasia or worse (CIN+) ;. Given the strong etiologic association between high-risk HPV infection and cervical cancer, high-risk HPV testing is considered an alternative for cytologybased cervical cancer screening. A review of seven randomized controlled trials (RCT) concluded that HPV testing in combination with cytology detected % more CIN+ lesions in the first screening round compared with cytology-based screening alone for women aged years and older (% CI:..) 0. In the second screening round, fewer CIN+ lesions were detected in the HPV arm than in the cytology arm (risk ratio 0. (% CI: 0. 0.)). The cumulative CIN+ detection rates over two screening rounds were not different (risk ratio.0 (% CI: 0..)) 0. The specificity of HPV DNA tests for CIN+ is low, and the positive predictive value (PPV) is low in groups with low CIN+ incidence (e.g. particularly young, sexually active women). The real cause of cervical cancer is not the HPV virus infection per se, but the continuous over-expression of the viral oncogenes E and E from oncogenic HPV types. There may be several reasons why E and E are over-expressed, but one of the main reasons is virus s integration into the human genome (loss of E gene results in loss of transcript regulation). The loss of the E gene only occurs in a small proportion of women with HPV infection. This implies that a test that detects the over-expression of E and E mrna is more specific than a test that detects the presence of viral DNA. HPV E/E mrna is a rational target for detecting HPV infections leading to cellular transformation. The HPV mrna test PreTect HPV-Proofer detects E/E mrna of the five main high-risk HPV types, namely,,,, and, which are associated with % of cervical cancers in Europe ;. Due to the higher specificity for CIN+ of PreTect HPV- Proofer compared to other HPV tests -, this test may have favorable characteristics in - BMJ Open: first published as 0./bmjopen--0 on August. Downloaded from on January by guest. Protected by copyright.

6 Page of BMJ Open 0 screening for cervical precancers. The aim of this study was to evaluate the performance of an HPV mrna test in primary cervical cancer screening. Material and methods The study comprised women years of age visiting selected gynecologists or general practitioner in Norway between May st 0 and December st 0. Women were included after an individual assessment by their physician. The departments of pathology and microbiology, University Hospital of Northern Norway, Tromsø, organized the study. Within the study s legal framework, participant identification from the unique -digit personal identification number, kept by all Norwegian citizens, was merged with lifetime data on cervical cytology and histology in four national registries administered by Norwegian Cancer Registry (NCR). With these mergers, a complete follow-up through December st 0 was possible. A mandatory reporting on cancer to NCR has been practiced since, on cervical cytology since (), and cervical histology since 0 from all cytology/pathology laboratories. In addition, since gynecology departments and practitioners collecting cervical biopsies/doing CIN treatment have been encouraged to report individual case-report-forms to NCR on CIN treatment In the merged dataset, we excluded women with an abnormal smear history (n= ) or a histology history of CIN+ (n=) prior to or until completed three months ( days) after the date of screening. In addition we excluded women aged < years (n= 0) or > years (n=). A total of women years in a situation resembling primary screening (Table ) were eligible Both conventional Pap tests and liquid-based cytology (LBC) were analyzed in local cytology laboratories. In cases with conventional Pap tests, extra specimen collections (PreTect TM) were made for HPV analysis. We extracted cervical cells from LBC by the ThinPrep 00 (Cytyc Corporation, Marlborough, MA, USA) for cytological examination. DNA/RNA was isolated from ml of the leftover material of the LBC (or from the PreTect TM). All women were subjected to detection of HPV mrna (PreTect HPV-Proofer, PreTect AS, Klokkarstua, Norway) in one laboratory. The HPV laboratory worked independently of the cytology departments, being unaware of the cytology results at time of HPV detection and vice versa. Biopsies were evaluated by pathologists, and histological results were reported using CIN terminology. Pathology laboratories in Norway use the same classification system for cytology (Bethesda) and histology (CIN). Every year all the laboratories receive reports from - BMJ Open: first published as 0./bmjopen--0 on August. Downloaded from on January by guest. Protected by copyright.

7 Page of 0 the NCR on laboratory-specific diagnostic performance. Annual meetings are held so cytologists and pathologist secure consistent classifications of diagnosis in cervical cancer prevention. The national screening program in Norway recommends all women aged years to have cervical cytology (Pap test) every third year. A national quality manual for cervical cancer prevention is continuously updated ( Women with normal cytology are encouraged to do another screen within three years. Women with highgrade cytology (ASC-H / HSIL) are referred immediately to colposcopy and biopsy. Prior to 0, women with equivocal or low-grade cytology (ASC-US / LSIL) were triaged by repeat cytology after six months. Women with repeated equivocal or low-grade cytology (ASC-US / LSIL x ) were triaged by repeat cytology after an additional months. Women with persistent equivocal or low-grade cytology (ASC-US / LSIL x ) over a period of months were referred to colposcopy and biopsy. Delayed HPV triage was implemented in the national screening program in 0. Women with ASC-US / LSIL were recommended repeat cytology and HPV testing after months. Women with positive HPV test and abnormal cytology were referred to colposcopy and biopsy. Women with positive HPV test and normal cytology were re-tested after months for persistence. Women with two consecutive positive HPV tests were referred for colposcopy and biopsy ( In Norway, the threshold for treatment by conization or LLETZ (large loop excision of the transformation zone) is CIN. Outcome assessment was based on the histological result of biopsies, where CIN+ was considered as the target disease and CIN and no CIN were considered as absence of disease. Only cervical cancer reported in the cancer file (validated by NCR against pathology reports at hospital) were true cases of cancer. SPSS version was used to conduct all statistical analyses, which entailed Chisquare tests, Mann-Whitney tests, and survival analyses. A p-value < 0.0 was considered statistically significant. The Regional Committee for Medical Research Ethics, Region East, Oslo, Norway, reviewed the study. The committee approved merging the laboratory data on HPV and cytology/histology data from NCR without informed consent from the participants (REK Sør- Øst 0/). - BMJ Open: first published as 0./bmjopen--0 on August. Downloaded from on January by guest. Protected by copyright.

8 Page of BMJ Open 0 Results At study start the detection rate of HPV mrna was.% (n=) among women with no history of abnormal smears nor history of abnormal cervical histology registered in NCR. Nearly half (/=.%) of the HPV-positive women were HPV--positive. HPV- was the single infection in of infections (.%) (Tables and ). HPV- was more prevalent than HPV-, HPV-, and HPV-. For all HPV types the infection prevalence decreased significantly by age (Tables and ) (p<0.00). The women were followed for women months, with a mean of. months and a range of 0 months. During the study period of 0 months, women (.%) developed CIN+. The distribution of CIN relative to CIN+ cases decreased by age (% among women years, % in women years). The cumulative proportion of both CIN+ (Table ) and CIN+ (Table /Figure ) were significantly higher in the younger ( years) than older ( years) women (survival analysis, p<0.00). At and months of follow-up, the cumulative proportions of CIN+ were 0.% (% CI: 0..) and.% (% CI:..), respectively, for women aged years, and 0.% (% CI: 0.-0.) and 0.% (% CI: 0.-.0), respectively, for women aged years. After adjusting for HPV status at baseline, there were no differences in proportions of CIN+ or CIN+ (Figure ) between HPV-negative and HPV-positive women aged or years. However, HPV-positive women had significantly higher proportions of both CIN+ and CIN+ independent of age. The slope of the curve for proportions of CIN+ (Figure ) among the younger HPV-positive women ( years at baseline) increased linearly after months, while the slope of the curve for proportions of CIN+ among older women ( years) leveled off (insignificant). The cumulative proportions of CIN+ and CIN+ (includes four cases of adenocarcinoma in situ) at and months are displayed in Table by HPV status at baseline. In total, the cumulative proportions of CIN+ or CIN+ were significantly higher in HPV- than HPV-/-/--positive women at baseline, whereas there were no differences in CIN+/CIN+ among HPV-- and HPV--positive women, or among HPV-- and HPV-/-/--positive women (small sample size) (Table and Figure ). The slope of the curves, as displayed in Figure, were similar across age (data not shown), but were inconsistently significant due to small numbers. The cumulative proportions of CIN+ and CIN+ among HPV-negative women remained low throughout the study period (Table and Figure ). - BMJ Open: first published as 0./bmjopen--0 on August. Downloaded from on January by guest. Protected by copyright.

9 Page of 0 All five cases of cervical cancer were diagnosed among women years or older. Case numbers and were years at baseline, with HPV-- and HPV--positive, respectively, After and months of observation, respectively, these two were diagnosed with squamous cell and adenosquamous carcinoma. The three other cases were at ages,, and years at baseline, were all HPV-negative, and were diagnosed after,, and months of observation two adenosquamous carcinomas and one squamous cell carcinoma. The cumulative incidence of cervical cancer was.0 (% CI:..) per women months at months of observation. - BMJ Open: first published as 0./bmjopen--0 on August. Downloaded from on January by guest. Protected by copyright.

10 Page of BMJ Open 0 Discussion Our study examines a low-risk population as we excluded at study start all women with a previous history of abnormal cytology and/or histology from cervix uteri. These exclusions removed persistent HPV infections expressing on and off low-grade lesions over time, thus reducing HPV positivity. In addition, HPV positivity will be lower with an mrna test targeting oncogenic expression, compared with a DNA test that detects HPV presence. The present HPV mrna positivity rate of.% in women years and.% in women years is much lower than reported from studies using HPV DNA tests. In the Horizon study, across all age groups, of, (.%) samples were HPV DNApositive (Cobas 00, Roche Molecular Systems, Pleasanton CA, USA) with normal cytology. This proportion decreased from.% in women aged years to.% at age years and.0% at age years. This implies that HPV DNA primary screening in women younger than years will lead to huge administrative challenges in light of low incidence of cervical cancer. The risks of CIN+ after months of follow-up were 0.% overall and 0.% in the HPV mrna-negatives, a relative reduction of %. The risks of CIN+ after months of follow-up were.% overall and 0.% in the HPV mrna-negatives, a relative reduction of %. Our results are in line with outcomes for primary screening with HPV DNA tests ;-. A Danish study using Hybrid Capture, CIN+ after months of follow-up reported.0% overall and.0% in the HPV DNA-negatives, a relative reduction of %. In a US cohort of 0 women, cytology/hpv women had a CIN+ cumulative incidence rate of 0.% after months and 0.% after months. Similarly, in a German cohort of 0 women, 0.% of cytology/hpv women developed CIN+ during five years of follow-up. In a Dutch cohort of 0 women, there was only one case of CIN+ among women with negative results on both tests during. years of follow-up. In a US cohort of women, one-time negative HR-HPV test at enrolment provided greater reassurance over the -year follow-up than a one-time negative Pap against CIN+ (.% versus.%) and CIN+ (0.0% versus.%). By comparison, cumulative incidence was.% for CIN+ and 0.% for CIN+ after one-time HPV and Pap tests that were both negative. In our study the cumulative incidence for CIN+ after six years was.% HPV-positive/cytology negative and 0.% for women with double negative Pap and HPV mrna test, respectively. As CIN+ incidence depends upon HPV prevalence, some heterogeneity between studies might be explained by differences in HPV prevalence and age at inclusion. - BMJ Open: first published as 0./bmjopen--0 on August. Downloaded from on January by guest. Protected by copyright.

11 Page 0 of 0 In a meta-analysis of European HPV primary studies, the cumulative incidence rate of CIN+ after six years was considerably lower among women with negative HPV at baseline (0.%, % CI: 0. 0.) than among women with negative results on cytology (0.%, % CI: 0..). By comparison, the cumulative incidence rate for women with negative cytology results at the most commonly recommended screening interval in Europe (three years) was 0.% (% CI: 0. 0.). The cumulative incidence rate among women with negative cytology results who were positive for HPV increased continuously over time, reaching 0% at six years, whereas the rate among women with positive cytology results who were negative for HPV remained below %. The HPV mrna test employed in our study detects five HPV types (,,,, and ) associated with % of cervical cancers in Europe ;. Women with other HPV types had a negative HPV mrna test. Still, the cumulative risk of CIN+ six years after a negative HPV mrna test was low (0.%). Most HPV DNA tests detect or HPV types. In a Danish study most of the HPV-positive CIN+ cases were of HPV-, -, -, and -. The cumulative risk of CIN+ after six years in women with a positive HPV test of other HPV types was not significantly different from women with a negative Hybrid Capture. In Europe, HPV predominates in both CIN and cervical cancer. Other HPV types have a slower progression into cancer. In countries with an organized cervical cancer screening program, the risk of cervical cancer development is higher for HPV types,,,, and than for other HPV types. These observations support the use of a specific HPV mrna test for detecting the five main HPV types in triage of women with minor cytological lesions. It is important to improve the screening in young women. CIN detection from years of age is important for decreasing the risk of cervical cancer, even in the postvaccinated era. In the ATHENA trial, more CIN+ was found in women than in all women and older. Of all CIN+ found in the study, % occurred among women, versus % in women years or older. Over the last years Norway has witnessed an increase in cervical cancer rates in women younger than years, despite cytology screening ( In the US corroborating data from the National Cancer Institute s SEER Tumor Registry show a sharp rise in the incidence of invasive cervical cancer between the ages of and years ( Table.). In the ATHENA study,.% of women years of age with CIN+ had a negative cytology (NILM). - BMJ Open: first published as 0./bmjopen--0 on August. Downloaded from on January by guest. Protected by copyright.

12 Page of BMJ Open 0 In 0/0 there were no national guidelines for HPV testing and follow-up of HPV-positive women. Most CIN+ cases were detected by passive follow-up in routine cervical cancer screening based on cytology every three years. This may result in verification bias favoring cytology when women with abnormal cytology were followed up, but not women with a positive HPV test. Cervical cytology has been the conventional method for cervical cancer screening in developed countries. Organized cytology-based screening programs have reduced the incidence of cervical cancer. However, cytological evaluation has a low clinical sensitivity ;. The method is subjective and has poor reproducibility. It is well established that HPV DNA testing is more sensitive for CIN+ than cytology -. Screening by HPV DNA tests may reduce the incidence of cervical cancer more than cytology screening ;;. A major challenge is the lower specificity and higher positivity rates when using HPV DNA testing in primary screening compared to cytology. The main cause of invasive cervical cancer is the deregulated and persistent production of HPV E and E oncoproteins. Hence, HPV E/E mrna is a rational target for detecting HPV infections leading to cellular transformation. In the present study we used an HPV mrna test that detects E/E mrna of the five main high-risk HPV types, namely,,,, and, found in cervical cancer. Given that HPV mrna-negative women retained their low risk of CIN+ for at least six years, frequent screening of these women may be unnecessary. New HPV infections are associated with an extremely low risk of cancer because they usually resolve without the need for medical intervention. Conclusion HPV mrna testing may be used as primary screening both for women years and years. Studies designed to do head-to-head comparison between HPV DNA and HPV mrna tests in primary screening of CIN+ are welcomed. - BMJ Open: first published as 0./bmjopen--0 on August. Downloaded from on January by guest. Protected by copyright.

13 Page of 0 Table legends Table. Selection of study population Table. Prevalence of HPV at study start by age Table. Cumulative proportion of CIN+ and CIN+ at and month by HPV status at baseline Figure legends Figure. Cumulative incidence of CIN+ by age Figure. Cumulative incidence of CIN+ in HPV-positive and HPV-negative women by age - BMJ Open: first published as 0./bmjopen--0 on August. Downloaded from on January by guest. Protected by copyright.

14 Page of BMJ Open 0 Table. Selection of study population N N Eligible History of ASCUS/LSIL History of HSIL History of CIN + Age < years Age >= 0 years CIN + at study start CIN + < days after study start In total Study population - BMJ Open: first published as 0./bmjopen--0 on August. Downloaded from on January by guest. Protected by copyright.

15 Page of 0 Table. Prevalence of HPV at study start by age Age (yrs) Total N HPV status % % % HPV-negative... HPV-/-/- (not, not ). 0.. HPV- (not ) HPV BMJ Open: first published as 0./bmjopen--0 on August. Downloaded from on January by guest. Protected by copyright.

16 Page of BMJ Open 0 Table. Cumulative proportion of CIN+ and CIN+ at and months by HPV status at baseline (% confidence intervals) months months CIN+ % CI CIN+ % CI HPV status N % % In total HPV-negative HPV-positive HPV-/-/- (not, not ) HPV- (not ) HPV CIN+ % CI CIN+ % CI HPV status N % % In total HPV-negative HPV-positive HPV-/-/- (not, not ) HPV- (not ) HPV BMJ Open: first published as 0./bmjopen--0 on August. Downloaded from on January by guest. Protected by copyright.

17 Page of 0 Contributorship statement Experiment conception and design: SWS SF TJG ESM FES. Experiment performance: SWS SF TJG ESM FES. Data analysis: SWS FES. Reagent/material/analysis tool contributions: SWS SF TJG ESM. Paper authors: SWS FES. All Authors read and approved the final manuscript. Competing interests The authors have declared that no competing interests exist. Funding This research was supported by the University Hospital of North Norway ( The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Data sharing statement The authors confirm that all data underlying the findings are fully available without restriction. Anonymous individual-level data and full dataset are available at the University Hospital of North Norway by ing corresponding author (sveinung.sorbye@unn.no). - BMJ Open: first published as 0./bmjopen--0 on August. Downloaded from on January by guest. Protected by copyright.

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21 Page of 0 () Mayrand MH, Duarte-Franco E, Rodrigues I, Walter SD, Hanley J, Ferenczy A et al. Human papillomavirus DNA versus Papanicolaou screening tests for cervical cancer. N Engl J Med 0; ():-. () Naucler P, Ryd W, Tornberg S, Strand A, Wadell G, Elfgren K et al. Human papillomavirus and Papanicolaou tests to screen for cervical cancer. N Engl J Med 0; ():-. () Ronco G, Giorgi-Rossi P, Carozzi F, Confortini M, Dalla PP, Del MA et al. Efficacy of human papillomavirus testing for the detection of invasive cervical cancers and cervical intraepithelial neoplasia: a randomised controlled trial. Lancet Oncol 0; ():-. () Schiffman M, Wentzensen N. Human papillomavirus infection and the multistage carcinogenesis of cervical cancer. Cancer Epidemiol Biomarkers Prev ; (): BMJ Open: first published as 0./bmjopen--0 on August. Downloaded from on January by guest. Protected by copyright.

22 Page of BMJ Open 0 Figure Cumulative incidence of CIN+ 0xmm (0 x 0 DPI) - BMJ Open: first published as 0./bmjopen--0 on August. Downloaded from on January by guest. Protected by copyright.

23 Page of 0 Figure Cumulative incidence of CIN+ 0xmm (0 x 0 DPI) - BMJ Open: first published as 0./bmjopen--0 on August. Downloaded from on January by guest. Protected by copyright.

24 Primary cervical cancer screening with an HPV mrna test a prospective cohort study Journal: BMJ Open Manuscript ID bmjopen--0.r Article Type: Research Date Submitted by the Author: -Jun- Complete List of Authors: Sørbye, Sveinung Wergeland; University Hospital of North Norway, Clinical Pathology Fismen, Silje; University Hospital of North Norway, Clinical Pathology Gutteberg, Tore; University Hospital of North Norway, Department of Microbiology and Infection Control; University of Tromsø, Department of Medical Biology, Faculty of Health Sciences Mortensen, Elin; University Hospital of North Norway, Clinical Pathology; University of Tromsø, Department of Medical Biology, Faculty of Health Sciences Skjeldestad, Finn Egil; UiT The Arctic University of Norway, Department of Community Medicine <b>primary Subject Heading</b>: Pathology Secondary Subject Heading: Epidemiology, Obstetrics and gynaecology, Public health, Sexual health Keywords: hpv mrna test, cervical intraepithelial neoplasia, CIN+, cervical cancer screening, hpv primary screening, hpv BMJ Open: first published as 0./bmjopen--0 on August. Downloaded from on January by guest. Protected by copyright.

25 Page of BMJ Open 0 0 Primary cervical cancer screening with an HPV mrna test a prospective cohort study Sveinung Wergeland Sørbye, Silje Fismen, Tore Jarl Gutteberg,, Elin Synnøve Mortensen,, Finn Egil Skjeldestad Department of Clinical Pathology, University Hospital of North Norway, Tromsø, Norway Department of Microbiology and Infection Control, University Hospital of North Norway, Tromsø, Norway Department of Medical Biology, Faculty of Health Sciences, University of Tromsø, Tromsø, Norway Institute of Community Medicine, Research Group Epidemiology of Chronic Diseases, UiT The Arctic University of Norway, Tromsø, Norway *Corresponding author and reprints, Sveinung Wergeland Sørbye, MD Department of Clinical Pathology University Hospital of North Norway 0 Tromsø, Norway Telephone, + or + 0 Fax, + 0 , sveinung.sorbye@unn.no - BMJ Open: first published as 0./bmjopen--0 on August. Downloaded from on January by guest. Protected by copyright.

26 Page of 0 Abstract Objectives: Assess the performance of -type HPV mrna test in primary screening within the framework of the Norwegian population-based screening program. Design: Nationwide register-based cohort study. Setting: In 0 0, general practitioners and gynecologists recruited women for participation in a primary screening study with a -type HPV mrna test. Participants: After excluding women with a history of abnormal smears and with CIN+ before or until months after screening, women aged years were eligible for study participation. The Norwegian Cancer Registry completed follow-up of CIN+ through December 0. Interventions: Follow-up according to the algorithm for cytology outcomes in the population-based Norwegian Cervical Cancer Screening Programme Main outcome measures: We estimated cumulative incidence of cervical intraepithelial neoplasia grade or worse (CIN+) months after -type HPV mrna test. Results:.% of the women were HPV mrna-positive at baseline. The overall cumulative rate of CIN+ was.% (% CI:..) through months of follow-up,.% for women years (n= ) and 0.% for women years (n= ). Cumulative CIN+ rates by baseline status for HPV mrna-positive and mrna-negative women years were.% (% CI:..) and 0.% (% CI: 0..), respectively, and.% (% CI: 0..) and 0.% (% CI: 0. 0.), respectively, in women years. Conclusions: The present cumulative incidence of CIN+ is similar to rates reported in screening studies via HPV DNA tests. Due to differences in biologic rationale and test characteristics, there is a trade-off between sensitivity and specificity that must be balanced when decisions on HPV tests in primary screening are taken. HPV mrna testing may be used as primary screening both for women years and years. - BMJ Open: first published as 0./bmjopen--0 on August. Downloaded from on January by guest. Protected by copyright.

27 Page of BMJ Open 0 Strengths and limitations of this study We consider studying primary screening with a -type HPV mrna test in a population of women with no previous CIN+ and/or abnormal smears as a strength as the HPV infections diagnosed are likely to be new infections. We consider the follow-up within the Norwegian Cervical Cancer Screening Programme as strength, as women regardless of mobility within Norway, are captures by the surveillance system for both cytology, histology and treatment. We consider just having one screening round with the -type HPV mrna test as a limitation, in addition to follow-up based on cytology only (verification bias). - BMJ Open: first published as 0./bmjopen--0 on August. Downloaded from on January by guest. Protected by copyright.

28 Page of Introduction Cervical cancer is the fourth most common cancer among women worldwide. Cervical human papillomavirus (HPV) infection is necessary but not sufficient to cause cervical cancer ;. HPV is one of the most common sexually transmitted infections with a prevalence ranging from to % in women years of age ;. The lifetime risk of HPV infection in women is 0 0%. Approximately HPV genotypes, referred to as high-risk (HR) types, are considered etiological agents of cervical cancer. HPV is by far the most significant HPV type in persisting infection and promoting progression into high-grade cervical intraepithelial neoplasia or worse (CIN+) ;. Given the strong etiologic association between high-risk HPV infection and cervical cancer, high-risk HPV testing is considered an alternative for cytologybased cervical cancer screening. A review of seven randomized controlled trials (RCT) concluded that HPV testing in combination with cytology detected % more CIN+ lesions in the first screening round compared with cytology-based screening alone for women aged years and older (% CI:..) 0. In the second screening round, fewer CIN+ lesions were detected in the HPV arm than in the cytology arm (risk ratio 0. (% CI: 0. 0.)). The cumulative CIN+ detection rates over two screening rounds were not different (risk ratio.0 (% CI: 0..)) 0. The specificity of HPV DNA tests for CIN+ is low, and the positive predictive value (PPV) is low in groups with low CIN+ incidence (e.g. particularly young, sexually active women). The real cause of cervical cancer is not the HPV virus infection per se, but the continuous over-expression of the viral oncogenes E and E from oncogenic HPV types. There may be several reasons why E and E are over-expressed, but one of the main reasons is virus s integration into the human genome (loss of E gene results in loss of transcript regulation). The loss of the E gene only occurs in a small proportion of women with HPV infection. This implies that a test that detects the over-expression of E and E mrna is more specific than a test that detects the presence of viral DNA. Not all cervical cancers have integrated viral forms; between % will have episomal forms only. Integration is not a requisite for transformation. HPV E/E mrna is a rational target for detecting HPV infections leading to cellular transformation. The HPV mrna test PreTect HPV-Proofer detects E/E mrna of the five main high-risk HPV types, namely,,,, and, which are associated with % of - BMJ Open: first published as 0./bmjopen--0 on August. Downloaded from on January by guest. Protected by copyright.

29 Page of BMJ Open cervical cancers in Europe ;. Due to the higher specificity for CIN+ of PreTect HPV- Proofer compared to other HPV tests -, this test may have favorable characteristics in screening for cervical precancers. The aim of this study was to evaluate the performance of an HPV mrna test in primary cervical cancer screening. Material and methods The study comprised women years of age visiting gynecologists and general practitioners in different parts of Norway between May st 0 and December st 0. Women were included after an individual assessment by their physician. The departments of pathology and microbiology, University Hospital of Northern Norway, Tromsø, organized the study. Within the study s legal framework, participant identification from the unique -digit personal identification number, kept by all Norwegian citizens, was merged with lifetime data on cervical cytology and histology in four national registries administered by Norwegian Cancer Registry (NCR). With these mergers, a complete follow-up through December st 0 was possible. A mandatory reporting on cancer to NCR has been practiced since, on cervical cytology since (), and cervical histology since 0 from all cytology/pathology laboratories. In addition, since gynecology departments and practitioners collecting cervical biopsies/doing CIN treatment have been encouraged to report individual case-report-forms to NCR on CIN treatment. In the merged dataset, we excluded women with an abnormal smear history (n= ) or a histology history of CIN+ (n=) prior to or until completed three months ( days) after the date of screening. In addition we excluded women aged < years (n= 0) or > years (n=). A total of women years in a situation resembling primary screening (Table ) were eligible. Both conventional Pap tests and liquid-based cytology (LBC) were analyzed in local cytology laboratories. In cases with conventional Pap tests, extra specimen collections (PreTect TM) were made for HPV analysis. We extracted cervical cells from LBC by the ThinPrep 00 (Cytyc Corporation, Marlborough, MA, USA) for cytological examination. DNA/RNA was isolated from ml of the leftover material of the LBC (or from the PreTect TM). All women were subjected to detection of HPV mrna (PreTect HPV-Proofer, PreTect AS, Klokkarstua, Norway) in one laboratory. The HPV laboratory worked independently of the cytology departments, being unaware of the cytology results at time of HPV detection and vice versa. - BMJ Open: first published as 0./bmjopen--0 on August. Downloaded from on January by guest. Protected by copyright.

30 Page of 0 Biopsies were evaluated by pathologists, and histological results were reported using CIN terminology. Pathology laboratories in Norway use the same classification system for cytology (Bethesda) and histology (CIN). Every year all the laboratories receive reports from the NCR on laboratory-specific diagnostic performance. Annual meetings are held so cytologists and pathologist secure consistent classifications of diagnosis in cervical cancer prevention. The national screening program in Norway recommends all women aged years to have cervical cytology (Pap test) every third year. A national quality manual for cervical cancer prevention is continuously updated ( Women with normal cytology are encouraged to do another screen within three years. Women with highgrade cytology (ASC-H / HSIL) are referred immediately to colposcopy and biopsy. Prior to 0, women with equivocal or low-grade cytology (ASC-US / LSIL) were triaged by repeat cytology after six months. Women with repeated equivocal or low-grade cytology (ASC-US / LSIL x ) were triaged by repeat cytology after an additional months. Women with persistent equivocal or low-grade cytology (ASC-US / LSIL x ) over a period of months were referred to colposcopy and biopsy. Delayed HPV triage was implemented in the national screening program in 0. Women with ASC-US / LSIL were recommended repeat cytology and HPV testing after months. Women with positive HPV test and abnormal cytology were referred to colposcopy and biopsy. Women with positive HPV test and normal cytology were re-tested after months for persistence. Women with two consecutive positive HPV tests were referred for colposcopy and biopsy ( The HPV tests used in Norway during 0 0 were Hybrid Capture II, PreTect HPV-Proofer and Amplicor. In Norway, the threshold for treatment by conization or LLETZ (large loop excision of the transformation zone) is CIN. Outcome assessment was based on the histological result of biopsies, where CIN+ was considered as the target disease and CIN and no CIN were considered as absence of disease. Only cervical cancer reported in the cancer file (validated by NCR against pathology reports at hospital) were true cases of cancer. SPSS version was used to conduct all statistical analyses, which entailed Chisquare tests, Mann-Whitney tests, and survival analyses. A p-value < 0.0 was considered statistically significant. The Regional Committee for Medical Research Ethics, Region East, Oslo, Norway, reviewed the study. The committee approved merging the laboratory data on HPV and - BMJ Open: first published as 0./bmjopen--0 on August. Downloaded from on January by guest. Protected by copyright.