Incidence of Cervical Cancer in HPV-positive Women With Low-grade Cytological Abnormalities
Conditions: Cervical Intraepithelial Neoplasia, Cervix Uteri Cancer, Human Papilloma Virus, Cervix Carcinoma, Cervix; Intraepithelial Neoplasia
Sponsor: Gynaecologisch Oncologisch Centrum Zuid

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PROTOCOL TITLE ‘Incidence of Cervical Cancer in HPV-positive Women with Low-grade 
Cytological Abnormalities’ 
Protocol ID 
2025-03 
Short title 
Incidence of Cervical Cancer in HPV-positive Women 
with Low-grade Cytological Abnormalities 
Version 
2.3 
Date 
November 2025 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

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TABLE OF CONTENTS  
1.INTRODUCTION AND RATIONALE................................................................................... 7 
2.OBJECTIVES ..................................................................................................................... 8 
3.STUDY DESIGN ................................................................................................................ 9 
4.STUDY POPULATION ....................................................................................................... 9 
4.1 Population (base) ......................................................................................................... 9 
4.2 Inclusion criteria ........................................................................................................... 9 
4.3 Exclusion criteria .......................................................................................................... 9 
5.METHODS ........................................................................................................................10 
5.1 Study parameters/endpoints .......................................................................................10 
5.1.1 Main study parameter/endpoint ............................................................................10 
5.1.2 Secondary study parameters/endpoints ................................................................10 
5.1.3 Other study parameters ........................................................................................10 
6.STATISTICAL ANALYSIS .................................................................................................11 
6.1 Primary study parameter(s) .........................................................................................11 
6.2 Secondary study parameter(s) ....................................................................................11 
6.3 Other study parameters ..............................................................................................11 
7.ETHICAL CONSIDERATIONS ..........................................................................................13 
7.1 Regulation statement ..................................................................................................13 
7.2 Recruitment and consent ............................................................................................13 
8.ADMINISTRATIVE ASPECTS, MONITORING AND PUBLICATION .................................14 
8.1 Handling and storage of data and documents .............................................................14 
8.2 Amendments ...............................................................................................................14 
8.3 Public disclosure and publication policy ......................................................................14 
9.REFERENCES ..................................................................................................................15 
 
 
 
 
 
 
 

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LIST OF ABBREVIATIONS AND RELEVANT DEFINITIONS  
AGC: Atypical Glandular Cells of Endocervical Origin 
ASC-US: Atypical Squamous Cells of Undetermined Significance 
CIN: Cervical Intraepithelial Neoplasia 
HPV: Human Papillomavirus 
IQR: Interquartile Range 
LSIL: Low-grade Squamous Intraepithelial Lesion 
NILM: Negative for Intraepithelial Lesion or Malignancy 
PALGA: Dutch nationwide pathology databank 
SD: Standard Deviation 
 
 

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SUMMARY 
Rationale: 
Since 2017, the Dutch cervical cancer screening program has adopted human papillomavirus 
(HPV) testing with referral for colposcopy in all HPV-positive women with cytological abnor-
malities. However, most low-grade abnormalities — atypical squamous cells of undetermined 
significance (ASC-US), atypical glandular cells of endocervical origin (AGC), and low-grade 
squamous intraepithelial lesions (LSIL) — regress spontaneously, which may result in overdi-
agnosis, overtreatment, and unnecessary pressure on the healthcare system. To evaluate 
whether follow-up intensity can be safely reduced, it is important to assess the actual cancer 
risk in this subgroup and compare it with the risk in women with stable negative for intraepithe-
lial lesion or malignancy (NILM) cytology after one year, who are currently returned to the 
screening program. 
Objective: 
• 
Primary objective: To determine the five-year incidence of histologically confirmed cer-
vical cancer in HPV-positive women with low-grade cytological abnormalities detected 
through the Dutch cervical cancer screening program. 
• 
Secondary objectives: 
o 
To assess time to cervical cancer diagnosis, all-cause mortality, the type of ex-
amination at which the diagnosis was established, and the histological charac-
teristics of the carcinoma (subtype) in this population, within five years after 
enrollment. 
o 
To assess cervical cancer risk stratified by histological diagnosis within three 
months of baseline abnormal cytology (≤ cervical intraepithelial neoplasia (CIN) 
I vs CIN 2+) and, for the ≤ CIN I group, further stratified by cytology at 12-month 
follow-up (no or nondiagnostic cytology, ≤ LSIL, HSIL+). 
o 
To perform a sub-analysis of women with a history of abnormal cytology or his-
tology and assess their five-year cervical cancer risk after enrollement. 
o 
To evaluate the incidence of cervical cancer in women with stable NILM cytol-
ogy after one year and compare it with the five-year incidence in women with 
low-grade cytological abnormalities. 

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Study design: 
Retrospective cohort study based on routinely collected data from the Dutch population-based 
cervical cancer screening program. 
Study population: 
Women who participated in the cervical cancer screening program in the Netherlands between 
January 2017 and December 2018, who: 
• 
Tested HPV-positive with cytology results categorized as ASC-US, AGC, or LSIL ac-
cording to the Bethesda system, or 
• 
Had stable NILM cytology after one year. 
Main study parameters/endpoints: 
Primary endpoint: Incidence of histologically confirmed cervical cancer within the five-year 
screening interval. 
Secondary endpoints: 
o 
Time to diagnosis of cervical cancer, all-cause mortality, the type of examination 
at which the diagnosis was established, and the histological characteristics of 
carcinoma (subtype), within five years after enrollment. 
o 
Cervical cancer incidence in women with a history of abnormal cytology or his-
tology. 
o 
Cervical cancer risk stratified by histological diagnosis within three months of 
baseline abnormal cytology (≤ CIN I vs CIN 2+) and, for the ≤ CIN I group, 
further stratified by cytology at 12-month follow-up (no or nondiagnostic cytol-
ogy, ≤ LSIL, HSIL +). With a follow-up period of five years and three months 
after enrollment.  
o 
Cervical cancer incidence in women with stable NILM cytology after one year. 
 
 

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Nature and extent of the burden and risks associated with participation, benefit and 
group relatedness: 
As this is a retrospective cohort study using existing screening data, no direct burden, risks, or 
interventions are associated with study participation. The potential benefit lies at the population 
level: results may provide evidence to optimize follow-up strategies for HPV-positive women 
with low-grade cytological abnormalities or persistent NILM results, and for those with relevant 
medical history, thereby reducing unnecessary procedures while maintaining safe cancer de-
tection. 
 
 

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1. INTRODUCTION AND RATIONALE 
Cervical cancer is the fourth most common cancer among women worldwide and remains a 
leading cause of cancer-related mortality.[1] Prevention is largely possible through organized 
screening programs. These typically involve the collection of cervical samples to test for the 
presence of human papillomavirus HPV and to detect cytological abnormalities suggestive of 
(pre)cancerous lesions.[2,3] Implementation of such screening programs has been associated 
with a reduction in the incidence of cervical cancer.[4,5] 
In the Netherlands, the national cervical cancer screening program invites women aged 30 to 
60 every five years.[6] Since 2017, the Dutch program has shifted from a cytology-based to  
primary HPV based screening, according to international guidelines.[6–8] This shift aims to 
improve early detection of high-grade lesions and reduce cervical cancer incidence, as over 
90% of women with cervical cancer are HPV-positive.[9] Women who test HPV-positive 
undergo follow-up cytology, and those with abnormal cytology—regardless of grade— are 
referred for colposcopy. 
However, a considerable proportion of HPV-positive women present with low-grade cytological 
abnormalities, which often regress spontaneously without progressing to cervical cancer [10–
12] Previous studies have shown that the long-term risk of cervical cancer in these women is 
relatively low. Nonetheless, referring them for colposcopy places a burden on healthcare 
resources, contributes to patient anxiety, and may lead to overtreatment.[13] In the context of 
rising healthcare costs and increasing pressure on the medical system, it is increasingly 
relevant to assess whether follow-up intensity for this subgroup can be safely reduced. 
To date, the long-term risk of cervical cancer in HPV-positive women with low-grade cytological 
abnormalities has not been thoroughly evaluated. Given that eligible women are invited for 
screening every five years, it is crucial to assess whether these baseline abnormalities may 
progress to cervical cancer within that interval. Therefore, this study investigates the five-year 
incidence of cervical cancer among HPV-positive women with low-grade cervical abnormalities 
participating in the Dutch cervical cancer screening program. 
 
 

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2. OBJECTIVES 
Primary Objective: 
To determine the five-year incidence of histologically confirmed cervical cancer in HPV-positive 
women with low-grade cytological abnormalities detected through the Dutch cervical cancer 
screening program. 
Secondary Objectives: 
• 
To assess time to cervical cancer diagnosis, all-cause mortality, type of examination at 
which the diagnosis was established and the histological characteristics of the 
carcinoma (subtype) in this population , within five years after enrollment. 
• 
To assess cervical cancer risk stratified by histological diagnosis within three months 
of baseline abnormal cytology (≤ CIN I vs CIN 2+) and, for the ≤ CIN I group, further 
stratified by cytology at 12-month follow-up (no or nondiagnostic cytology, ≤ LSIL, HSIL 
+), with a follow-up period of five years and three months after enrollment. 
• 
To perform a sub-analysis of women with a history of abnormal cytology or histology 
and assess their five-year cervical cancer risk. 
• 
To evaluate the incidence of cervical cancer in women with stable NILM cytology after 
one year and compare it to the five-year incidence in women with low-grade cytological 
abnormalities.  
 
 

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3. STUDY DESIGN 
This retrospective cohort study includes women who participated in the Dutch national cervical 
cancer screening program between January 2017 and December 2018, with a follow-up period 
of five years. 
4. STUDY POPULATION 
4.1 Population (base) 
The study population consists of women who participated in the Dutch national cervical cancer 
screening program between January 2017 and December 2018. Eligible women were HPV-
positive at baseline with either low-grade cytological abnormalities (ASC-US, AGC, LSIL) or 
stable NILM cytology one year after initial screening. 
4.2 Inclusion criteria 
To be eligible to participate in this study, a participant must meet the following criteria: 
• 
Women participating in the Dutch national cervical cancer screening program between 
January 2017 and December 2018. 
• 
HPV-positive at baseline screening. 
• 
One of the following cytology results: 
o 
ASC-US, AGC, or LSIL cytology according to the Bethesda system 
or 
o 
NILM cytology at one-year follow-up. 
4.3 Exclusion criteria 
There are no exclusion criteria. 
 
 

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5. METHODS 
5.1 Study parameters/endpoints 
5.1.1 Main study parameter/endpoint 
• 
Incidence of histologically confirmed cervical cancer within the five-year screening in-
terval in HPV-positive women with low-grade cytological abnormalities. 
5.1.2 Secondary study parameters/endpoints 
• 
Time to diagnosis of cervical cancer. 
• 
All-cause mortality. 
• 
Type of examination at which cervical cancer diagnosis was established. 
• 
Histological characteristics of carcinoma (subtype). 
• 
Cervical cancer incidence in women with stable NILM cytology after one year. 
• 
Cervical cancer incidence in women with a history of abnormal cytology or histology. 
• 
Cervical cancer risk stratified by histological diagnosis within three months of baseline 
abnormal cytology (≤ CIN I vs > CIN I) and, for the ≤ CIN I group, further stratified by 
cytology at 12-month follow-up (no or nondiagnostic cytology, ≤ LSIL, > LSIL). 
5.1.3 Other study parameters 
• 
Baseline characteristics such as age at inclusion. 
• 
Cytology according to the Bethesda system and histology results at baseline and fol-
low-up. 
• 
HPV test results at baseline and follow-up. 
• 
Relevant medical history, including prior abnormal cytology or histology. 
 
 

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6. STATISTICAL ANALYSIS 
6.1 Primary study parameter(s) 
The primary study parameter is the incidence of histologically confirmed cervical cancer within 
the five-year follow-up period. The cumulative incidence will be calculated as the number of 
new cases diagnosed divided by the total number of women included in the cohort. The inci-
dence rate will also be calculated as the number of cervical cancer cases divided by the total 
follow-up time in person-years and expressed per 1,000 person-years. 
6.2 Secondary study parameter(s) 
Secondary study parameters include: 
• 
Time-to-diagnosis of cervical cancer, which will be displayed in a line diagram with time 
on the y-axis and number of cases on the x-axis. 
• 
All-cause mortality, type of examination at which the diagnosis was established, and 
histological subtype of cervical cancer, which will be summarized as frequencies and 
percentages for each relevant group. 
• 
Cervical cancer incidence in women withstable NILM cytology after one-year follow-up, 
cervical cancer incidence in women with a history of abnormal cytology or histology, 
and risk stratification by histology within three months of baseline cytology and follow-
up cytology at 12 months. For these parameters, cumulative incidence and incidence 
rates will be calculated.  
6.3 Other study parameters 
Other study parameters include baseline characteristics such as age, which will be assessed 
for normality using the Shapiro-Wilk test. If normally distributed, this variable will be reported 
as means with standard deviation (SD). If non-normally distributed, it will be reported as medi-
ans with interquartile range (IQR). Cytology and histology results at baseline and follow-up, 
HPV test results, and relevant medical history will be summarized as frequencies and percent-
ages for each relevant group. For the relevant medical history, the highest histology results will 
be presented. In case a patient has both cytological and histological results, histology will be 
prioritized. Pap scores will be converted to Bethesda system classifications using the KOPAC 
scores.[14] Missing data will be described, and analyses will include all available data. 

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All statistical analyses will be conducted using SPSS Statistics version 29 (IBM Corp., Armonk, 
NY, USA, 2025). 
 
 

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7. ETHICAL CONSIDERATIONS 
7.1 Regulation statement 
This study will be conducted in accordance with the principles of the Declaration of Helsinki 
(2013 version) and in compliance with applicable Dutch laws and regulations. The study was 
approved by the scientific committee of Palga, the Dutch nationwide pathology databank in the 
Netherlands (approval number 2025-3). Ethical approval by a medical ethical committee was 
not required under Dutch law, as no patients were directly involved in the conduct of the re-
search and only non-identifiable data were used.  
7.2 Recruitment and consent 
No recruitment or informed consent procedures were applicable, as the study uses retrospec-
tively collected, fully anonymized data from Palga.  
 
 

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8. ADMINISTRATIVE ASPECTS, MONITORING AND PUBLICATION 
8.1 Handling and storage of data and documents 
Data for this study are obtained from Palga (the Dutch nationwide pathology databank). All 
data and results will be used solely for the purposes described in this study and will not be 
used for any other purpose or published beyond this scope. The data will not be shared with 
third parties not involved in the research. Data will be stored securely and will be destroyed 
five years after publication. 
All data are fully anonymized. No procedures will be performed that could reveal the identity 
of participants if indirectly identifying information is inadvertently included. Access to the data 
is restricted to the research team. Procedures are in place to ensure data security and partici-
pant privacy. 
8.2 Amendments  
Amendments are changes made to the research after a favourable opinion by the review 
committee has been given. All amendments will be notified to the review committee that gave 
a favourable opinion. Non-substantial amendments will not be notified to the review committee, 
but will be recorded and filed by the sponsor.  
8.3 Public disclosure and publication policy 
The results of the study will be published in peer-reviewed scientific journals and presented at 
scientific meetings.  
 
 

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9. REFERENCES 
[1] 
Arbyn M, Weiderpass E, Bruni L, Sanjosé S de, Saraiya M, Ferlay J, et al. Estimates of 
incidence and mortality of cervical cancer in 2018: a worldwide analysis. The Lancet 
Global Health 2020;8:e191–203. https://doi.org/10.1016/S2214-109X(19)30482-6. 
[2] 
Bruni L, Serrano B, Roura E, Alemany L, Cowan M, Herrero R, et al. Cervical cancer 
screening programmes and age-specific coverage estimates for 202 countries and 
territories worldwide: a review and synthetic analysis. The Lancet Global Health 
2022;10:e1115–27. https://doi.org/10.1016/S2214-109X(22)00241-8. 
[3] 
US Preventive Services Task Force. Screening for Cervical Cancer: US Preventive 
Services Task Force Recommendation Statement. JAMA 2018;320:674–86. 
https://doi.org/10.1001/jama.2018.10897. 
[4] 
Melnikow J, Henderson JT, Burda BU, Senger CA, Durbin S, Soulsby MA. Screening 
for Cervical Cancer With High-Risk Human Papillomavirus Testing: A Systematic 
Evidence Review for the U.S. Preventive Services Task Force. Rockville (MD): Agency 
for Healthcare Research and Quality (US); 2018. 
[5] 
Jansen EEL, Zielonke N, Gini A, Anttila A, Segnan N, Vokó Z, et al. Effect of organised 
cervical cancer screening on cervical cancer mortality in Europe: a systematic review. 
Eur J Cancer 2020;127:207–23. https://doi.org/10.1016/j.ejca.2019.12.013. 
[6] 
Cervical cancer screening programme. RIVM n.d. https://www.rivm.nl/en/cervical-
cancer-screening-programme (accessed June 1, 2025). 
[7] 
WHO guideline for screening and treatment of cervical pre-cancer lesions for cervical 
cancer prevention n.d. https://www.who.int/publications/i/item/9789240030824 
(accessed June 1, 2025). 
[8] 
Eramus MC, Palga. Bevolkingsonderzoek Baarmoederhalskanker - Monitor 2017. RIVM 
2017. 
[9] 
Bosch FX, Manos MM, Muñoz N, Sherman M, Jansen AM, Peto J, et al. Prevalence of 
human papillomavirus in cervical cancer: a worldwide perspective. International 
biological study on cervical cancer (IBSCC) Study Group. J Natl Cancer Inst 
1995;87:796–802. https://doi.org/10.1093/jnci/87.11.796. 
[10] Ciavattini A, Serri M, Di Giuseppe J, Liverani CA, Gardella B, Papiccio M, et al. Long-
term observational approach in women with histological diagnosis of cervical low-grade 
squamous intraepithelial lesion: an Italian multicentric retrospective cohort study. BMJ 
Open 2019;9:e024920. https://doi.org/10.1136/bmjopen-2018-024920. 
[11] Katki HA, Gage JC, Schiffman M, Castle PE, Fetterman B, Poitras NE, et al. Follow-up 
testing after colposcopy: five-year risk of CIN 2+ after a colposcopic diagnosis of CIN 1 
or less. J Low Genit Tract Dis 2013;17:S69-77. 
https://doi.org/10.1097/LGT.0b013e31828543b1. 
[12] The Netherlands: Integraal Kankercentrum Nederland. National Guidelines: CIN, AIS en 
VAIN (Dutch). Richtlijnendatabase Federatie Medisch Specialisten;Accessed June 
2025. 
[13] Sharp L, Cotton S, Cruickshank M, Gray NM, Harrild K, Smart L, et al. The unintended 
consequences of cervical screening: distress in women undergoing cytologic 
surveillance. J Low Genit Tract Dis 2014;18:142–50. 
https://doi.org/10.1097/LGT.0b013e31829c97d8. 
[14] The Netherlands: Integraal Kankercentrum Nederland. National Guidelines: 
Cervixcytologie- Codering van de uitslag (Dutch). Richtlijnendatabase Federatie 
Medisch Specialisten; Accessed June 2025.