Ongoing projects

Functional characterization of microRNAs and their mRNA targets regulating HPV-associated anchorage-independent growth.

Funding: Dutch Cancer Society (KWF 2018-11312)

PIs: dr. Renske Steenbergen, dr. Renée de Menezes.

Anchorage dependence of epithelial cells (keratinocytes) refers to the need of a stable surface for cell growth. Cancer cells are capable of growing in the absence of anchorage dependence, and so-called anchorage-independent growth represents a critical hallmark of cancer. If epithelial cells turn into anchorage-independent cells during malignant transformation, these cells represent the ultimate precancerous stage before invasive cancer. At present little is known about the molecular events driving acquisition of anchorage independence in epithelial cells and more basic insight into the molecular origin of this phenomenon is warranted.
This project aims to apply functional genomics to characterize miRNAs and their mRNA targets involved in acquisition of anchorage-independent growth using a unique in vitro model system of HPV-containing keratinocytes.
By a genome-wide miRNA mimic screen on UltraLASC-plates using anchorage-dependent and anchorage-independent passages of HPV-transfected keratinocytes we aim for the identification of both oncogenic and tumor-suppressive miRNAs associated with anchorage independence.
The biologically relevant mRNA targets will be identified by a CRISPR-Cas9 knock-out screen for predicted mRNA targets of miRNAs. Screen data will be analysed at the Epidemiology and Biostatistics department (dr. Renee de Menezes). Functional validation includes soft-agar assays, the current gold standard for anchorage independence and luciferase reporter assays to study miRNA-mRNA interactions.
In this project, we will obtain important basic insights into the molecular origin of anchorage-independent growth, which particularly reflects the transition of precancerous lesions to invasive cancer. The miRNAs and mRNA targets found to be involved in the acquisition of anchorage independence in epithelial cells will provide diagnostic biomarkers for early detection, i.e. at the stage of precancerous disease at which clinical intervention is essential to prevent invasive cancer ("less cancer''), and predictive biomarkers to guide treatment ("more cure").



DNA methylation biomarkers in cervical cancer screening: towards all-molecular screening with self sampling.

Funding: Dutch Cancer Society (KWF 2018-11337)

PIs: dr. Daniëlle Heideman, dr. Maaike Bleeker, dr. Hans Berkhof

Advances in cervical screening have recently been implemented, including an improved molecular screening tool testing for the presence of HPV, the causative agent of cervical cancer, and self-sampling to attract more women into screening. These modalities, however, gave rise to novel challenges, including the need for triage to identify only those HPV-positive women with clinically relevant disease in need of treatment. Triage is currently performed by cytology which requires repeat testing and cannot reliably be performed on self-samples. This highlights the need for improved biomarkers for cervical cancer screening.
Cervical cancer development following a persistent infection with high-risk HPV is driven by additional host cell changes, such as altered DNA methylation. DNA methylation biomarkers provide an intriguing new molecular tool for cervical screening. Our recent studies show that DNA methylation biomarkers may well complement HPV-based screening, being compatible with both physician-taken and self-collected samples. The aim of this project is to extensively validate and further develop a DNA methylation biomarker panel as a tool for HPV-triage and primary screening that is fully compatible with self-sampling.


Cancer risk assessment of anal intraepithelial neoplasia using DNA methylation markers

Funding: Dutch Cancer Society (KWF 2016-10781)

PIs: dr. Renske Steenbergen, prof. dr. Jan Prins (AMC), prof.dr. Henry de Vries (AMC).

Anal cancer is an increasing problem in HIV+ MSM. Like cervical cancer, anal cancer is caused by oncogenic HPV types, and preceded by precursor lesions: high-grade anal intraepithelial neoplasia (HGAIN). Currently patients are screened by high-resolution anoscopy (HRA) plus biopsies, a complicated, costly and cumbersome procedure. HGAIN is found in over 30% of HIV+ MSM, and only a minority of HGAIN lesions will progress to cancer, yet all HGAIN are currently treated. Treatment of HGAIN is difficult, painful and has suboptimal response rates and high recurrence rates. Consequently, screening and treatment often needs to be repeated. Ideally, only HGAIN with high malignant potential (advanced HGAIN) are treated in a more aggressive fashion. Yet, up to now there are no clinicopathological parameters to predict the progression to anal cancer. Hence, there is an urgent need to 1) identify men at risk of having advanced HGAIN or anal cancer needing HRA diagnostics and subsequent aggressive treatment; 2) prevent over-referral and overtreatment of HIV+ MSM with a low cancer risk
as a screening tool in swabs to detect anal cancer and HGAIN with a high cancer progression risk.
Our recent studies show that DNA methylation analysis of tumour suppressor genes in cervical swabs provides a promising tool to detect cervical cancers and high-grade cervical intraepithelial neoplasia with a high short term cancer progression risk (advanced HGCIN). Given the similar pathogenesis of cervical and anal cancer DNA methylation markers might likewise be a valuable tool to detect advanced HGAIN.
This project aims to 1) validate the diagnostic value of methylation markers to identify anal cancer and HGAIN lesions with a high cancer progression risk (advanced HGAIN), and 2) determine their utility as screening tool in anal swabs, thereby enabling tailored diagnostics and management of anal dysplasia in HIV+ MSM.


RNA modifying enzymes diversify the microRNA repertoire and function during early stage cancer development

Funding: Dutch Cancer Society (KWF 2016-10395)

PIs: dr. Renske Steenbergen, dr. Michiel Pegtel, dr. Daniela Koppers-Lalic.

MicroRNAs (miRNAs) are 20-21 nucleotide non-coding RNAs that regulate gene expression and show consistent deregulation in cancer. We observed that severe miRNA deregulation occurs early in carcinogenesis, which could only partly be explained by (epi)genomic alterations. We comprehensively studied miRNA deregulation with next-generation sequencing (RNA-Seq) and demonstrate in vivo and in vitro that miRNAs in precancerous cells exist as multiple length variants called isomiRs. Strikingly their distribution seems to change dramatically during early transformation. IsomiRs comprise many different modifications, including elongations, trimmings, editing and non-templated additions (NTAs). These modifications are generated by RNA-modifying enzymes that include ribonucleotidyl transferases (rNTAses). In silico analysis suggest that these enzymes are altered in cancer, possibly exerting a hereto non-described oncogenic activity. In this project we will address the potential role of rNTAses in early carcinogenesis in a well-established model system. using a unique and well-characterized in vitro model of HPV-induced carcinogenesis. This study will result in detailed knowledge on the role of rNTAses in the diversification of key miRNAs in cancer development, which is an important next step to expedite diagnostic implementation of miRNA-derived isomiRs in cancer care.

Cancer risk assessment in women with vulvar intraepithelial neoplasia

Funding: Dutch Cancer Society (KWF 2016-10382)

PIs: dr. Maaike Bleeker, Prof.dr. Hans Berkhof, dr. Renske Steenbergen, dr. Marc van Beurden 

Vulvar intraepithelial neoplasia (VIN) is often caused by human papillomavirus (HPV) infection and frequently presents as multifocal vulvar disease in young women. The incidence of VIN is rising and a possible reduction by prophylactic HPV vaccination is only to be expected after at least three decades. Only a small subset of women with VIN (<10%) will progress to vulvar squamous cell carcinoma (VSCC) but clinical and histological classifications are insufficient to predict the cancer risk. Affected women frequently have to undergo multiple mutilating surgical interventions associated with psychosexual morbidity. This mutilating over-treatment in women with VIN can largely be prevented when their clinical care would be guided by biomarkers reflecting the cancer progression risk. Preliminary molecular analyses on VIN and VSCC, showed an increase in both genetic and epigenetic (i.e. DNA methylation) changes with progression of VIN to VSCC. VIN of women with associated VSCC (VIN with VSCC) displayed more chromosomal aberrations and higher methylation levels of tumor suppressor genes compared to VIN of women who did not develop VSCC during follow-up (VIN without VSCC). In this project we aim to further assess and validate the clinical value of methylation markers for cancer risk stratification in women with VIN.

Preventing overtreatment in CIN2/3 lesions: The role of methylation markers in predicting (non-) regression

Funding: ZON-MW, The Netherlands Organization for Health Research and Development

PIs: prof.dr. Peter Snijders, prof.dr. Gemma Kenter

Current cytology-based cervical screening programmes serve to detect and treat high-grade precursor lesions (CIN2/3) to prevent cervical cancer. However, the diagnostic-treatment trajectory is associated with considerable overtreatment since CIN2/3 lesions, particularly in young women, have a high spontaneous regression rate. Pathologists are unable to differentiate between CIN2/3 lesions with a low short-term progression risk to cervical cancer (productive lesions), not in need of immediate treatment, and those with a high short-term progression risk (transforming lesions) that need immediate treatment. Individual cancer risk prediction of CIN2/3 is therefore essential to reduce overtreatment. Recently, it has been shown that DNA methylation markers can differentiate between productive and transforming CIN2/3. Here, we aim to validate prospectively that testing for the methylation status of a CIN2/3 predicts (non-) regression leading to prevention of overtreatment.


Molecular Self Screening for Cervical Cancer Prevention: Discovery and validation of DNA methylation miRNA markers for triage of women with hrHPV positive self-samples.

Funding FP7-IDEAS-ERC European Research Council Advanced Mass-care (322986)

PI: prof.dr. Chris Meijer

Objective: Cervical carcinoma is a preventable cancer caused by a persistent infection with high-risk types of human papillomavirus (hrHPV). Several western countries have implemented nation-wide screening programs based on the detection of abnormal cells in cervical smears (cytology) for cervical cancer prevention. Although these programs have markedly reduced incidence in developed countries, even the most sophisticated cytology-based screening programs miss significant numbers of high-grade cervical lesions and fail to further reduce cervical cancer incidence. Besides loss to follow-up of women with abnormal cytology, the major causes underlying failure are: 1) Low sensitivity of the primary cytology based screening test, which is subjective, often not or poorly quality assessed (QA) and has low sensitivity for precancerous lesions, 2) Incomplete coverage of women invited to participate in the screening program. Remarkably, in developed countries with a successful nation-wide cervical screening program, 55% of all carcinomas are diagnosed in women who do not participate. 3) The lack of QA cytology in medium and low resource countries is one of the main reasons why incidence and mortality stay high in these countries.
In recognition of these shortcomings the MASS-CARE project envisions a novel, objective, full molecular self-screening approach that enables molecular screening and triage for referral to the gynaecologist using self-collected cervicovaginal samples as an alternative tool for cervical cancer screening. In particular, we aim to discover molecular triage markers, based on DNA methylation and miRNA expression, that are specific for the stratification of women with hrHPV positive self-samples. The research team headed by professor Meijer has a strong track record of cutting edge translational research, has state-of-the art molecular tools in place and is equipped with unique biobanks of clinical cohorts of thousands of hrHPV positive women that are needed to successfully execute this ambitious project.

Comparing Health Services interventions for the prevention of HPV-related cancer (CoheaHr).

Funding EU FP7-Health

PIs: prof.dr. Hans Berkhof, prof.dr. Peter Snijders, prof.dr Chris Meijer

Objective: Important progress has been made in the field of HPV-disease prevention with the development and implementation of HPV vaccines and HPV DNA screening. In the CoheaHr project, the (cost-) effectiveness of different European preventive strategies will be compared. The goal is to build a reliable and comparable evidence base on the (cost-) effectiveness of these policies implemented under country-specific preventive services conditions. To achieve this goal, a set of specific tasks will be carried out. Three randomized trials will be performed in organised screening settings to determine: i) whether self-collection of specimens for HPV DNA testing is an effective and feasible alternative for physician-based sampling, ii) whether screening intervals can be extended in women vaccinated at young age, iii) whether vaccinating women two years before entering the screening programme will favour the use of HPV screening. The first and third randomized trials are multi-country trials whereas the second trial will be carried out in a cohort of Finnish women vaccinated in 2007. For unvaccinated, 25-45 year old women participating in screening, acceptability and general feasibility of HPV vaccination will be studied in a multi-country demonstration survey. Comparisons by transmission models are included to provide long-term projections for cancer incidence and mortality. Furthermore, the establishment of a standardised joint European data warehouse will be continued and extended for (continuous) evaluation of comparative effectiveness of screening and vaccination policies in Europe. Finally, there will be an ongoing effort for producing systematic reviews and meta-analyses which provide a sustainable resource for evidence. CoheaHr will provide a strong evidence base which enable policy and other decision makers to make informed decisionmaking on HPV prevention strategies, thereby contributing to strengthening health systems and health services interventions in Europe.


MIMICS: MicroRNA Isoforms for Molecular Interception of Cervical cancer using Self-samples

Funding: ERC Proof-of-Concept grant 2015

PIs: prof.dr Chris Meijer, dr. Renske Steenbergen

The ERC frontier program Mass-Care project (ERC advanced grant) aims at the development of full molecular self-screening for cervical cancer as an alternative to current cytology-based cervical cancer screening. In Mass-Care, we investigate the value of two classes of molecular markers, DNA methylation markers and microRNA (miRNA) markers for the detection of cervical (pre) cancer. In the process of our analyses, we frequently observed variations at the 3’ end of the 11 discovered miRNA markers. Such variations are called isomiRs, and can have great impact on their detectability by standard PCR-based assays and could thus constitute a great improvement compared to using the canonical miRNA sequence.

In this ERC-PoC project, we will bring the innovation of using isomiRs for cervical (pre)cancer screening to proof-of-concept. By analysing the potential relationship between these isomiRs and cervical disease in detail, we aim to select the isomiRs that reveal the strongest association with cervical cancer and could have additional value as disease biomarker compared to the canonical counterparts of respective miRNAs. The identified isomiRs, which improve cervical cancer detection compared to the canonical miRNAs will be added to the panel of DNA methylation and canonical miRNAs as detected in the ERC-advanced project. This assay will constitute the first full molecular, non-morphology-based, objective screening assay for cervical cancer fully compatible with self-sampling.

Prevention of overdiagnosis and overtreatment of HPV-positive women by methylation marker testing.

Funding KWF VU2014-7238

PIs: dr. Daniëlle Heideman, prof.dr. Peter Snijders, prof.dr. Chris Meijer

Dutch summary: Secundaire preventie van baarmoederhalskanker (BMHK) kan verbeterd worden door uitstrijkjes van vrouwen te testen op de aanwezigheid van het humaan papillomavirus (HPV), de veroorzaker van BMHK. De HPV test zal in 2016 in het Nederlandse bevolkingsonderzoek BMHK worden ingevoerd. Deze test biedt tevens mogelijkheden voor zelf-afname van materiaal voor onderzoek; een belangrijke methode om meer vrouwen te screenen.
Echter, een nadeel van de HPV test is dat deze ook HPV infecties van voorbijgaande aard meet en de daaraan gekoppelde voorloper (cervicale intraepitheliale neoplasie; CIN) afwijkingen, die nooit zullen ontaarden in BMHK. In de praktijk leidt dit tot overdiagnose en overbehandeling, tenzij een vervolgtest wordt gebruikt die op aanwezigheid van klinisch relevante CIN afwijkingen test. Om over-verwijzing van HPV-positieve vrouwen naar de gynaecoloog te verminderen, zal in het toekomstige Nederlandse bevolkingsonderzoek aanvullende cytologie (Pap test) worden uitgevoerd. Echter, cytologie is subjectief en een herhaaltest na 6 maanden is nodig om een voldoende gevoeligheid te bereiken. In de praktijk komt echter 25-40% van de vrouwen niet meer opdagen voor zo’n herhaaltest. Daarnaast detecteert cytologie ook vroege CIN afwijkingen die niet zullen ontaarden in BMHK.
Verder is een aanzienlijk aantal vrouwen die bij de gynaecoloog komt vanwege klachten, jonger dan 30 jaar. In deze jonge vrouwen komen voorbijgaande HPV infecties, veelal gepaard gaande met afwijkende cytologie, vaak voor, terwijl BMHK zeldzaam is en de meeste CIN afwijkingen in deze leeftijdscategorie spontaan verdwijnen. Behandeling van CIN afwijkingen kan gepaard gaan met aanzienlijke morbiditeit, zoals moeilijker zwanger worden, insufficiëntie van de baarmoedermond en vroeggeboorte.
Dus, er is een sterke behoefte aan biomarkers die kunnen bepalen welke HPV-positieve vrouwen BMHK hebben óf ernstige CIN afwijkingen, die op korte termijn in BMHK ontaarden.
Onze recent ontdekte biomarker FAM19A4, gebaseerd op DNA methylering, is in dit kader zeer veelbelovend. Uit voorwerk blijkt dat FAM19A4 methyleringsanalyse een zeer hoge (dwz. 100%) gevoeligheid heeft voor BMHK en de meest ernstige CIN afwijkingen, die behandeling nodig hebben.
In het hier voorgestelde project willen wij de klinische waarde van deze biomarker verder uittesten, gebruikmakende van prospectief verzameld materiaal van unieke cohorten van HPV-positieve vrouwen. Hierbij zullen de gevoeligheid en specificiteit van de FAM19A4 methyleringstest voor BMHK en ernstige CIN afwijkingen worden onderzocht in zowel een bevolkingsonderzoekpopulatie (vrouwen ouder dan 30 jaar) als een populatie vrouwen die de polikliniek gynaecologie bezoekt (zowel vrouwen jonger als ouder dan 30 jaar) in relatie tot de cytologietest, het materiaaltype (arts- en zelf-afgenomen materiaal), en leeftijd.
Dit onderzoek zal een gevalideerde FAM19A4 methyleringstest opleveren die toepasbaar is op zowel huisarts- als zelf-afgenomen materiaal, om het vervolgbeleid van HPV-positieve vrouwen te bepalen. De test zal resulteren in minder ongerustheid onder HPV-positieve vrouwen, het aantal onnodige verwijzingen naar de gynaecoloog verminderen en overdiagnose/overbehandeling reduceren, daar waar een hoge beschermingsgraad tegen BMHK blijft bestaan.


Construction and clinical validation of a miRNA marker panel for triage in HPV-based cervical cancer screening.

Funding KWF VU 2012-5708


PIs: dr. Renske Steenbergen, dr. Saskia Wilting, prof.dr. Peter Snijders

Dutch summary: Baarmoederhalskanker wordt veroorzaakt door een infectie met hoog-risico types van het humaan papillomavirus (HPV). Echter, naast HPV zijn ook (epi)genetische veranderingen in het genoom van de geïnfecteerde cel noodzakelijk voor de ontwikkeling van baarmoederhalskanker. Het huidige bevolkingsonderzoek voor baarmoederhalskanker is gebaseerd op de detectie van afwijkende cellen in een uitstrijkje, ook wel cytologie genoemd. Eerder onderzoek heeft aangetoond dat de effectiviteit van het huidige bevolkingsonderzoek verbeterd kan worden door 1) eerst te testen op de aanwezigheid van HPV en 2) niet-deelnemende vrouwen de mogelijkheid te bieden tot zelf-afname van cervico-vaginaal materiaal dat ook op aanwezigheid van HPV getest kan worden. De Gezondheidsraad heeft in mei 2011 geadviseerd beide aanpassingen door te voeren in het huidige bevolkingsonderzoek.
Aangezien maar een klein deel van de HPV infecties tot baarmoederhalskanker leidt, kan het bevolkingsonderzoek nog verder verbeterd worden door toevoeging van markers gebaseerd op de additionele genetische veranderingen die betrokken zijn bij baarmoederhalskanker. Zo kan bij een positieve HPV test beter voorspeld worden hoe hoog het risico op ontwikkeling van baarmoederhalskanker is en of directe doorverwijzing naar de gynaecoloog wel of niet noodzakelijk is. De recent ontdekte microRNAs (miRNAs) lijken in dit kader zeer veelbelovend. miRNAs zijn korte RNA moleculen die zelf niet coderen voor eiwitmoleculen, maar wel de expressie van andere eiwitten kunnen beïnvloeden. Er wordt geschat dat 1 miRNA de expressie van circa 200 eiwitten kan reguleren, waaronder eiwitten die kanker kunnen veroorzaken (onco- en tumor suppressor eiwitten). Eerdere studies hebben aangetoond dat miRNAs al in een vroege fase van het ontstaan van kanker betrokken zijn en bovendien een specifiek expressiepatroon hebben in baarmoederhalskanker en de bijbehorende ernstige voorloperlaesies. Daarnaast zijn miRNAs zeer stabiel waardoor ze direct gemeten kunnen worden in kleine hoeveelheden klinisch materiaal, zoals uitstrijkjes en zelf-afgenomen cervico-vaginaal materiaal.
In deze studie willen wij onderzoeken of afwijkende miRNA expressie zoals we die gevonden hebben in baarmoederhalskanker en de ernstige voorloperlaesies ook gedetecteerd kan worden in uitstrijkjes van vrouwen met een onderliggende ernstige laesie. Deze miRNAs worden vervolgens in een gevalideerd en al langer bestaand in vitro model getest om te bepalen of de miRNAs direct functioneel betrokken zijn bij het ontstaan van baarmoederhalskanker. Uiteindelijk zal deze studie resulteren in een gevoelig en specifiek marker panel van miRNAs die direct betrokken zijn bij het ontstaan van baarmoederhalskanker en waarvan de afwijkende expressie meetbaar is in uitstrijkjes. Dit panel zal kunnen worden toegevoegd aan het vernieuwde bevolkingsonderzoek en resulteren in een betere detectie van baarmoederhalskanker en voorloperlaesies. Niet alleen zal dit leiden tot minder ongerustheid bij de HPV-positieve vrouwen, ook zal het aantal onnodige doorverwijzingen naar de huisarts of gynaecoloog verminderen. Volledig moleculaire en daardoor objectieve screening voor baarmoederhalskanker biedt ook nieuwe mogelijkheden voor ontwikkelingslanden waar momenteel niet gescreend wordt.