Project: Cervix care: biomarker-based triage diagnostics for HPV positive women to detect cervical cancer

Invasive cervical cancer is the second-most common cancer among women worldwide with 585 278 incident cases and 327 899 cases of attributable deaths in 2010 (Ferlay, 2010). It is widely acknowledged that cervical cancer can be most-effectively prevented by screening. Therefore, many countries have implemented large screening programs that have been proven to substantially reduce the incidence and mortality of cervical cancer up to 80% (Arbyn et al 2009). _x000D__x000D_Currently, cervical cytology (or Pap smears) is still the cornerstone in community-based screening. The Pap test requires a cervical scraping, which has to be made by a physician. Women with unequivocally abnormal Pap smears are referred to a gynaecologist for colposcopy. When abnormalities are seen by colposcopy, biopsies of suspicious areas are taken for confirmation by histology. Women with equivocal cytological abnormalities are kept under surveillance. Histological grading of preinvasive, so-called CIN lesions is based on assessing the proportion of the epithelium occupied by dysplastic cells (CIN grading from 1 to 3). These cells are considered precancerous. CIN 1 lesions regress in most cases. CIN2 and CIN3 lesions (high-grade CIN) can progress into cervical cancer and are therefore treated in clinical practice. _x000D__x000D_Despite the value of these programs, it is expected that the incidence and mortality rates in the developed world will reCO constant primarily because the effect of screening has been largely realized in the past 30 years (Figure 1 addendum; Datamonitor, 2010). There are several factors that explain the failure of screening programs to further reduce the incidence and mortality of cervical cancer. The false–negative results of the Pap test, particularly for precursor lesions of cervical adenocarcinoma, the subjectivity of the Pap test (readout), and, perhaps most importantly, under-screening of the population at risk are among the many reasons for this failure. _x000D__x000D_Recent studies have demonstrated that testing for high-risk HPV (hrHPV), representing the CO causative agent of cervical cancer (Walboomers et al. 1999; Munoz et al. 2003), is more sensitive (although less specific) than the Pap test in detecting high-grade CIN (Cuzick et al., 2008). HPV testing provides therefore a valuable alternative compared to Pap testing in the detection hrHPV subtypes. Recent randomized-controlled cervical-cancer screening trials have shown that hrHPV testing yields a superior protection against cervical precancerous lesions and cervical cancer compared to Pap testing (Bulkmans et al. 2007; Ronco et al. 2010). Consequently, hrHPV testing is a more attractive primary cervical cancer screening tool than cytology, provided that the test does not detect too many clinically irrelevant, transient hrHPV infections that are typically characterized but low viral copy numbers (Meijer et al. 2010). _x000D__x000D_The lower specificity for CIN2 or worse (CIN2+) of HPV testing compared to Pap testing would lead to many false-positives (i.e. hrHPV positive women without disease). The consequence would be too many undesired adverse effects in the generally healthy population, such as anxiety and overtreatment. Furthermore, unnecessary high costs of the screening program would be a result (Rijkaart et al., 2011). To control the number of colposcopy referrals, hrHPV positive women should not be offered colposcopy immediately but be first further stratified by means of triage testing to distinguish hrHPV-positive women with CIN2+ lesions or cervical cancer in need of colposcopy from those without meaningful cervical disease (Rijkaart et al., 2011). Triage testing is therefore of utmost importance for large-scale introduction of HPV testing, preventing any undesired effects of false-positives. _x000D__x000D_At present, cytology is considered an appropriate triage tool for hrHPV-positive women. Immunostaining of cytology slides for p16INK4a with or without Ki-67 staining potentially yields better results than cytology. However, given their subjective nature, cytology-based methods are not fully compatible with the advantages of hrHPV testing in terms of reproducibility, quality, and sample flexibility, the latter hampering their performance on self-collected cervico-vaginal samples. _x000D__x000D_Based on cutting-edge research of Self-Screen BV, unique disease-related biomarkers (CADM1 & MAL) were discovered and patented. Through a combination with proprietary MSP methylation technology of MDx Health, both Ps join forces to develop a hypersensitive and specific triage test for women with hrHPV. By combining the efforts of MDx Health and Self-screen, the triage assay will be further developed and offered to the market as complete triage kit with CE and PMA approval. This development would allow worldwide large-scale introduction of primary HPV testing with triage testing coupled thereto, thereby significantly improving the efficacy of preventive screening programs. _x000D_

Acronym	Cervix-care (Reference Number: 6679)
Duration	01/09/2011 - 30/08/2013
Project Topic	High-risk HPV testing is more sensitive but less specific than the Pap test for detecting high-grade cervical disease. To prevent over referral, we aim to develop a unique, disease-related molecular triage test.
Project Results (after finalisation)	The project has resulted in the development and completion of a multiplex qMSP kit (referred to as the PreCursor-M kit) for triage of high-risk HPV positive women. CE-certifcation of the kit is nearly completed. The kit is based on the combined DNA hypermethylation analysis by quantitative methylation specific PCR (qMSP) of three tumor suppressor genes (CADM1, MAL and miR-124-2) and an internal DNA quality control (B-actin) in a single reaction. Major advantages of the multiplex assay are less hands-on time and the requirement of less amounts of DNA compared to conventional singleplex assays. Moreover data quality is improved by normalization of the three methylation markers to the reference gene present in the same reaction and the use of a internal calibrator. _x000D_The Precursor-M kit has been clinically validated on both cervical scrapes and self-collected cervico-vaginal lavage specimens of women testing positive for high-risk HPV DNA. The PreCursor-M kit is intended to be used for further clinical stratification of women with hrHPV positive samples, identified by an hrHPV screening assay fully clinically validated for the identification of women at risk for developing cervical cancer.
Network	Eurostars
Call	Eurostars Cut-Off 6

Project partner

Number	Name	Role	Country
2	MDx Health S.A.	Partner	Belgium
2	Self-screen BV	Coordinator	Netherlands