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  • The study reported here had the primary

    2020-10-15

    The study reported here had the primary goal to compare the methylation of HPV late genes with methylation of the DAPK promoter, and with histological or cytological diagnoses among high-risk patients that were referred to a colposcopy clinic based on abnormal cervical cytology. Based on the literature cited above, we considered DAPK the most promising among the cellular epigenetic markers and we intended to compare this diagnosis with that of the viral late gene methylation. Aside from HPV16 and HPV18, our study targeted HPV31 and HPV45, which had not yet been studied when this research was done, but has been reported since then (Wentzensen et al., 2012). Our research targeted the promoter region of the DAPK gene, and two or three amplicons of the L2 and L1 genes of the four high-risk HPV types.
    Results
    Discussion Our discovery of HPV16 and 18 methylation and their change during cervical carcinogenesis (Kalantari et al., 2004, Badal et al., 2004, Turan et al., 2006) have become generally confirmed and led to a major expansion of the available data Torin1 (Brandsma et al., 2009, Fernandez et al., 2009, Sun et al., 2011, Clarke et al., 2012, Mirabello et al., 2012a, Lorincz et al., 2013) and extension of this phenomenon to HPV31 and 45 (Wentzensen et al., 2012). It is now generally accepted that methylation is increased in high-grade lesions and cancer compared to asymptomatic infection or low-grade lesions, and that this mechanisms targets the late genes more than the early genes or the long control region. The question of the underlying mechanism has not become revisited after our reports of correlation between methylation and recombination between HPV genomes and cellular DNA (Kalantari et al., 2008a, Kalantari et al., 2008b, Kalantari et al., 2010), suggesting that transcriptionally inactive parts of the genome may become part of the heterochromatin like most exogenous DNA without relevance of the identity of the HPV genes (Doerfler et al., 2001). In contrast, endogenous tumor suppressor genes may be targeted at random by cellular de novo methylation, and cells with tumor suppressor genes inactivated by methylation may expand in number due the phenotypic consequences of the gene inactivation. While HPV methylation and cellular gene methylation are likely enzymatically related, the underlying mechanistic logic is clearly different. Our research adds new aspects to the epigenetic profiling of HPV lesions by concomitantly analyzing the methylation of the late genes of four high-risk HPV-types HPV16, 18, 31, and 45 and that of a cellular gene, DAPK, one of the best cellular biomarkers for the progression of cervical cancer. It is also unique by including a substantial number of cervical cancers beyond the study of high-grade precursor lesions. Our study confirms that methylation of L2/L1 occurs in all four HPV types, and increases between all five pathological categories, asymptomatic infection, ASCUS, LSIL/CIN1, HSIL/CIN2-3 and cervical cancer, the biggest increases occurring in the two progression steps between LSIL/CIN1 and cancer. Interestingly, a similar gain of methylation occurs in the methylation of DAPK, in contrast to the data from others (Sun et al., 2011). Our investigation is a pilot study of the potential of these biomarkers, as the numbers of samples, especially for HPV18, 31, and 45, is low. The value of biomarkers that are fully developed in invasive cancer and much less prevalent in patients with low-grade and high-grade lesion is at this point still debatable. It is our hypothesis that highly methylated LSIL/CIN1 and HSIL/CIN2-3 samples are those that have molecularly undergone changes that predestine them to develop into cancers. But the answer to the question of whether “high methylation” in general and which level of methylation in details identifies cells with an irrevocable propensity to grow into invasive cancer can only be resolved by future studies, in spite of support by other labs for the predictive value of HPV16 methylation (Mirabello et al., 2012b, Lorincz et al., 2013). The hypothesis needs further epidemiological and molecular evaluation. Epidemiological research could take the form of retrospective longitudinal studies, and ask whether cancer patients showed higher HPV L2/L1 and DAPK methylation in archival precancerous samples. Molecular research could reveal that the methylation is a mechanistic part of the etiological process. For example, as the normal life cycle of HPVs is irrevocably terminated by chromosomal recombination, methylation of the L2/L1 genes ensuing from recombination likely correlates with an interruption of E2 gene transcription and increased E6/E7 oncogene expression. On the other side, methylation of cellular tumor suppressor genes such as DAPK may suppress functions relevant for the maintenance of the non-cancerous state. We suggest that subsequent to such epidemiological and molecular confirmations the combination methylation analyses of HPV L2/L1 and cellular genes like DAPK and technical improvements by next-generation-sequencing will lead to clinically useful tests of early detection of cervical cancer progression.