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Validation of PreCursor-M for enhanced Cervical (Pre)Cancer detection

Periodic Reporting for period 3 - VALID-SCREEN (Validation of PreCursor-M for enhanced Cervical (Pre)Cancer detection)

Reporting period: 2017-07-01 to 2019-03-31

Cervical cancer (CC) is the fourth most common cancer in women (570.000 new cases 2018). CC is preventable with a screening program, and curable if found and treated early. In many European countries, cytology-based screening programs have resulted in a decrease in CC incidence. However, the effects have leveled off due to insufficient sensitivity of cytology, asking for innovations to the screening instruments. CC is caused by a persistent infection with a high-risk human papillomavirus (HPV) and women can be screened for the presence of a cervical hrHPV infection. Since testing for hrHPV DNA gives better protection against CC, HPV testing is being implemented as primary screening instrument in several European countries. A downside of the HPV test is that besides clinically meaningful infections also many transient HPV infections are detected resulting in a low specificity. Therefore, triage of HPV-positive women to identify women with cervical disease is essential to maintain a cost-effective screening program. The use of an effective triage test reduces overdiagnosis and unnecessary follow-up of women with transient infections. Additional (epi)genetic alterations are required for progression from precancer to invasive CC upon a persistent hrHPV infection. DNA hypermethylation of promoter regions of host-cell genes is a frequent alteration in cervical carcinogenesis. Aberrant DNA methylation thereby provides important biomarkers that can serve as a triage test for HPV-positive women to identify those at risk of CC. Self-screen has developed a CE-IVD methylation assay consisting of two biomarkers combined with a reference in one multiplex assay (FAM19A4/miR124-2 methylation assay). This assay detects CC with high accuracy. Precursor lesions that are detected by this methylation test have a cancer-like methylation-high profile and a high short term risk for progression to CC. Supplementing HPV screening with this methylation assay enables physicians to assess whether the HPV infection is associated with CC or precursor lesions in need of treatment. Thus, this methylation test provides clinicians guidance in the choice of follow up strategies, thereby reducing overreferral for colposcopy and overtreatment. During the course of the Valid-screen project, the FAM19A4/miR124-2 methylation assay was commercially launched as the QIAsure Methylation Test (2016) and its clinical performance was validated in different European cohorts of HPV-positive women. The project furthermore tackled important market implementation aspects. Altogether, the study provided the necessary proof of the effectiveness of the FAM19A4/miR124-2 methylation assay as a triage test and had been essential for the successful commercialization and market uptake of the QIAsure Methylation Test. A key diagnostic tool in the CC detection area to advance women’s health has been delivered.
The clinical performance of the FAM19A4/miR124-2 methylation assay (QIAsure Methylation Test) in different European countries was validated via the partnerships with various European institutes. The QIAsure Methylation Test performs robust and reproducible with good intra- and inter-laboratory agreement in the different laboratory contexts [Floore et al. Clin Lab Anal. 2019]. The QIAsure Methylation Test detects nearly all CC (>98%), with consistently high positivity rates across different CC histotypes [Vink et al. IJC 2019]. Over 4,600 cervical scrapes were analyzed with the QIAsure Methylation Test showing CIN3+ sensitivity of 78.6% at specificity of 73.4% in HPV-positive women over 30 years of age. Figures were 60.4% and 78.0%, respectively, in HPV-positive women below 30 years of age. These data support the use of the QIAsure Methylation Test for full molecular screening of CC, including primary HPV testing and triage testing by methylation analysis. The QIAsure Methylation Test is compatible with both clinician-collected and self-collected specimens, providing a full molecular self-screening option for women without visiting the general practitioner. In strategic partnering with QIAGEN, market implementation and co-education of clinicians and customers was further established. Satellite symposia dedicated to the QIAsure Methylation Test were held at the Eurogin congress of 2017 and 2018. The laboratory workflow was streamlined for high-throughput testing in diagnostic and screening laboratories. Triage algorithms for the QIAsure Methylation Test were developed, showing benefit over cytology as triage. Altogether, the Valid-screen project gained essential clinical performance data on the QIAsure Methylation Test and commercialized this new tool for CC screening. The data support the use of the QIAsure Methylation Test to distinguish the subgroup of HPV-positive women having a high risk of cervical (pre)cancer in need of further gynecologic examination. As such, the Valid-screen project has advanced the development of the QIAsure Methylation Test from TRL8 (system complete and qualified) to TRL9 (actual system proven in operational environment).
Precursor lesions of CC, named cervical intraepithelial neoplasia (CIN), are classically divided into CIN1, CIN2, and CIN3, of which CIN3 is considered the most advanced precursor. Common practice is that all CIN2 and CIN3 are treated to prevent CC development. However, it is well known in the field that many of these CIN2 and some of CIN3 spontaneously regress, making treatment actually unwarranted. Current CIN classification is not able to identify which lesions will progress. The present treatment approach thereby leads to considerable overtreatment and cervical morbidity, including frequent preterm birth and cervical insufficiency in women at fertile age.
DNA methylation analysis of genes involved in CC development represents an important change in the concept of treatment of CIN2/3. A positive methylation test reflects a cancer-like methylation-high pattern. This methylation pattern can be used as a biomarker for the detection of advanced lesions, defined as CIN2/3 lesions associated with a duration of HPV infection >5years and having many chromosomal alterations. These advanced CIN lesions (i.e. part of the CIN2 and most CIN3 lesions) have a high short-term risk of progression to CC and are in need of treatment [Steenbergen et al. Nat Rev Cancer 2014]. This concept fits well with knowledge in the field that CIN2/3 lesions are heterogeneous, both in clinical behavior and with respect to (epi)genetic alterations. For instance, most CIN2/3 lesions at young age (<30 years) will not develop into cancer and mainly regress. This means that the methylation-negative CIN2 and CIN3 lesions, which are detected by current cytology and/or HPV testing, are in fact early lesions with a low chance of progression and may not need treatment within 1-3 years. The use of DNA methylation analysis can reduce overdiagnosis and overtreatment, which is especially important for women at fertile age. To translate this new view - which can be seen as a paradigm shift in the treatment of CIN lesions - to profound benefits to women and for the economics of healthcare, the Valid-screen project has played an important role. The project enabled the collaboration with several international European laboratory sites with well-documented clinical specimens to perform the necessary clinical validation studies to support the new concept and enforce the paradigm shift.
Molecular cervical screening