Final Report Summary - MASS-CARE (Molecular Self Screening for Cervical Cancer Prevention)
Testing for the presence of high-risk human papillomavirus (HPV), the causative agent of cervical cancer, will become the preferred method for secondary prevention of cervical cancer. HPV testing can reliably be applied to both physician-taken and self-collected samples, the latter offering opportunities to increase screening compliance. A drawback, however, is the detection of transient HPV infections and associated cervical intraepithelial neoplasia (CIN) lesions that will not develop into cancer. This asks for a proper triage test to distinguish HPV positive women in need of treatment. The MASS-CARE project aimed at the development of an innovative screening strategy for cervical (pre)cancer based on women-friendly self-collection using full molecular examination.
Towards this goal, a discovery of new molecular markers able to discriminate between HPV-positive self-samples of women with and without cervical (pre)cancerous disease, with subsequent validations thereof, was performed in this project. Based on previous research we focused on two types of molecular markers: 1) Methylation markers, previously shown to be functionally involved in the carcinogenic process and found to be reliably detectable in self-sampled material, and 2) miRNA markers, described as key players in (cervical) oncogenesis and also easily detectable due to their extreme stability. During the first phase of this project we have generated genome-wide methylation and miRNA profiles of a selected series of self-samples from HPV-positive women with and without cervical (pre)cancerous disease. Innovative statistical methodology was developed and applied to these profiles to identify the most promising panels of methylation and miRNA markers, respectively.
A total of 12 methylation markers was selected and validated by an independent technique (qMSP) in self-samples, cell line models and tissue specimens of cervical (pre)cancerous lesions. Subsequent independent clinical validation on a large series of self-samples yielded a 3-gene methylation classifier with an excellent clinical performance to discriminate between healthy controls and women with CIN3 in both lavage and brush self-samples (AUC=0.90/0.86). It is intended to develop this 3-gene classifier into a diagnostic assay in cooperation with an industrial partner.
Additionally a total of 9 miRNA markers were selected and six were validated using RT-qPCR as an independent technique. By analysis of an independent series of lavage self-samples an optimal classifier consisting of 6 miRNAs was built, which showed a good clinical performance to discriminate between healthy controls and women with CIN3 and cervical cancer in lavage self-samples (AUC=0.72).
Although combined analysis showed that combination of methylation and miRNA markers increased the clinical performance, at present the development of a combined marker assay is a technically challenging task and asks for further investments.
In addition to the foreseen objectives, novel developments included the development of a novel statistical tool (GRidge) for genome-wide data analysis obtained from impure samples like self-samples. These methods are currently widely applied to other biomarker discovery studies. The miRNA validation studies revealed that for reliable miRNA expression analysis using qPCR adequate data normalization is essential to remove non-biological, technical variations. To this end a strategy of data normalization was developed and validated.
Moreover, based on the results obtained for the miRNA markers and the recent knowledge that more in depth analysis into miRs variants, so-called isomiRs, will be an important next step to prepare miRNAs for diagnostic application, we decided to first invest in further data analysis and technical developments and to save our precious clinical materials for validation of a further optimized test protocol and miRNA panel. These challenges in miRNA analysis are currently addressed in the project ERC-PoC-2015_713303 MIMICS.
In conclusion, the MASS-CARE project resulted in highly effective DNA methylation and miRNA based classifiers for direct triage on self-samples from HPV-positive women. The miRNA markers are most promising based on their high stability and further technical developments are needed to develop a test and proceed with clinical validation studies. The methylation markers on the other hand are ready to be developed into a diagnostic assay in collaboration with an industrial partner. Following further independent clinical validation in prospective studies and at external research institutes involving several European key opinion leaders these markers are expected to enable full molecular examination of cervico-vaginal self-samples and thereby reduce the worldwide cervical cancer burden.
Towards this goal, a discovery of new molecular markers able to discriminate between HPV-positive self-samples of women with and without cervical (pre)cancerous disease, with subsequent validations thereof, was performed in this project. Based on previous research we focused on two types of molecular markers: 1) Methylation markers, previously shown to be functionally involved in the carcinogenic process and found to be reliably detectable in self-sampled material, and 2) miRNA markers, described as key players in (cervical) oncogenesis and also easily detectable due to their extreme stability. During the first phase of this project we have generated genome-wide methylation and miRNA profiles of a selected series of self-samples from HPV-positive women with and without cervical (pre)cancerous disease. Innovative statistical methodology was developed and applied to these profiles to identify the most promising panels of methylation and miRNA markers, respectively.
A total of 12 methylation markers was selected and validated by an independent technique (qMSP) in self-samples, cell line models and tissue specimens of cervical (pre)cancerous lesions. Subsequent independent clinical validation on a large series of self-samples yielded a 3-gene methylation classifier with an excellent clinical performance to discriminate between healthy controls and women with CIN3 in both lavage and brush self-samples (AUC=0.90/0.86). It is intended to develop this 3-gene classifier into a diagnostic assay in cooperation with an industrial partner.
Additionally a total of 9 miRNA markers were selected and six were validated using RT-qPCR as an independent technique. By analysis of an independent series of lavage self-samples an optimal classifier consisting of 6 miRNAs was built, which showed a good clinical performance to discriminate between healthy controls and women with CIN3 and cervical cancer in lavage self-samples (AUC=0.72).
Although combined analysis showed that combination of methylation and miRNA markers increased the clinical performance, at present the development of a combined marker assay is a technically challenging task and asks for further investments.
In addition to the foreseen objectives, novel developments included the development of a novel statistical tool (GRidge) for genome-wide data analysis obtained from impure samples like self-samples. These methods are currently widely applied to other biomarker discovery studies. The miRNA validation studies revealed that for reliable miRNA expression analysis using qPCR adequate data normalization is essential to remove non-biological, technical variations. To this end a strategy of data normalization was developed and validated.
Moreover, based on the results obtained for the miRNA markers and the recent knowledge that more in depth analysis into miRs variants, so-called isomiRs, will be an important next step to prepare miRNAs for diagnostic application, we decided to first invest in further data analysis and technical developments and to save our precious clinical materials for validation of a further optimized test protocol and miRNA panel. These challenges in miRNA analysis are currently addressed in the project ERC-PoC-2015_713303 MIMICS.
In conclusion, the MASS-CARE project resulted in highly effective DNA methylation and miRNA based classifiers for direct triage on self-samples from HPV-positive women. The miRNA markers are most promising based on their high stability and further technical developments are needed to develop a test and proceed with clinical validation studies. The methylation markers on the other hand are ready to be developed into a diagnostic assay in collaboration with an industrial partner. Following further independent clinical validation in prospective studies and at external research institutes involving several European key opinion leaders these markers are expected to enable full molecular examination of cervico-vaginal self-samples and thereby reduce the worldwide cervical cancer burden.