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  • Altogether we have shown the


    Altogether we have shown the presence of fully methylated DAPK fragments in serum of ovarian cancer patients but also of women with uterine leiomyoma. This indicates the prerequisite for the analysis of not only healthy controls but also of women with other frequent non-cancer disease for the validation of methylation markers in serum. Due to the presence of hypermethylated DAPK in malignancies of different entities such as bladder, lung, colorectum, cervix uteri, SIS3 and lymphocytes [29], [31], [32], [33] this finding is of general interest for serum-based analyses.
    Conflict of interest statement
    Introduction Cervical cancer, premalignant cervical lesions and non-neoplastic HPV infections, i.e. atypical cells of undetermined significance (ASCUS) and cervical intraepithelial neoplasia (CIN), are diagnosed by cytology (Papanicolaou test, Pap test), colposcopic inspection, and histological examination of biopsies. These tests and procedures are successful at decreasing the incidence of cervical cancer, but their rate of false diagnoses is a matter of concern (Nanda et al., 2000, Stoler and Schiffman, 2001). Detection of the DNA of high-risk human papillomavirus (HPV) types (Munoz et al., 2003, Bernard et al., 2010), the primary cause of cervical cancer, has become a powerful criterion to amend these procedures, and has greatly increased the sensitivity of screening (Bulkmans et al., 2007, Mayrand et al., 2007, Naucler et al., 2007). However, since the fraction of women being infected by HPVs at some time of their lives (>80%) vastly exceed the incidence rate of cervical cancer (about 1%), and since a positive HPV DNA test often indicates a transient infection rather than a developing cervical cancer, HPV DNA diagnosis alone is not sufficient to distinguish women with benign infections from those requiring intensive management. In order to prevent unnecessary procedures on patients with abnormal Pap smears who are not at risk for developing cervical cancer, gynecologic practice needs tests that are sensitive and specific to detect high-risk patients. Numerous attempts have been made to measure markers that change as the result of HPV-dependent carcinogenesis, but these tests are still of limited benefit (von Knebel Doeberitz, 2002). The molecular mechanisms involved in the progression of asymptomatic or low-grade HPV infections to cervical cancer are yet poorly understood, but include the methylation of many of those cellular genes that are also epigenetically affected in cancers of other organ sites and without an HPV etiology. The search of clinically useful epigenetic biomarkers of cervical cancer that may allow risk stratification in patients began relatively recently, but this field of research expanded rapidly, and a review (Wentzensen et al., 2009) compared studies of more than 60 cellular genes. Unfortunately, this meta-analysis came to the conclusion that there is currently no single methylation marker that that has the appropriate performance to serve as cervical cancer biomarker. The reviewed studies point only to few genes, notably DAPK (death associated protein kinase 1) and RARB (retinoic acid receptor beta), which might be attractive targets of further evaluations. Notably, these two markers stood out in a large epidemiological study comparing a panel of twenty cellular methylation targets (Feng et al., 2005). Independently of these studies of cellular genes, our group has investigated how methylation affects HPV genomes in different stages of cervical neoplastic disease (Kalantari et al., 2004, Kalantari et al., 2008a, Kalantari et al., 2010, Badal et al., 2004, Turan et al., 2006, Turan et al., 2007), and our findings have been confirmed and expanded by others (Brandsma et al., 2009, Fernandez et al., 2009, Sun et al., 2011, Clarke et al., 2012, Mirabello et al., 2012a). A recent review summarizes this field (Johannsen and Lambert, 2013). Methylation of HPV16 and 18 increases among viral infections progressing from asymptomatic infection through low-grade and high-grade disease and malignancy. This effect is particularly pronounced in the late genes L2 and L1, whose products are not required for neoplastic processes. Methylation may affect the whole viral genome, however, although methylation is a repression mechanism (Bird, 2002). This is possible since neoplastic cells normally contain numerous HPV genomes. As long as one single HPV genome is spared from methylation, it maintains the carcinogenic process, although the other HPV genomes in the same cell may be transcriptionally silenced by methylation (Van Tine et al., 2004). The exact trigger of HPV methylation is not well understood, but there is evidence that methylation correlates with recombination between the HPV genome and chromosomal DNA (Kalantari et al., 2008a, Kalantari et al., 2008b, Kalantari et al., 2010). Studies not related to methylation have shown that HPV genomes frequently integrate into the cellular DNA in cancer, but it is disputed whether this mechanism is only a frequent event or mechanistically necessary (Daniel et al., 1997, Ueda et al., 2003, Hudelist et al., 2004, Arias-Pulido et al., 2006, Kulmala et al., 2006, Briolat et al., 2007, Pett and Coleman, 2007, Häfner et al., 2008, Vinokurova et al., 2008, Campitelli et al., 2012, Xu et al., 2013). Foreign DNA that integrates into mammalian chromosomal DNA is known to be a preferred methylation target, and therefore a correlation between HPV recombination and HPV DNA methylation may have nothing to do with the properties of the HPV genome and the biology of the virus (Doerfler et al., 2001). There is evidence that integration of HPV genomes favors the carcinogenic process as it leads to increased E6 and E7 oncoproteins transcription by interference with negative feedback by E2 proteins (Tan et al., 1994); transcriptional induction by the nuclear matrix (Stünkel et al., 2000), and stabilized E6/E7 transcripts (Jeon et al., 1995, Häfner et al., 2008).