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Cervical neoplasia-specific biomarkers, e.g. DNA methylation markers, with high sensitivity and specificity are urgently needed to improve current population-based screening on (pre)malignant cervical neoplasia. We aimed to identify new cervical neoplasia-specific DNA methylation markers and to design and validate a methylation marker panel for triage of high-risk human papillomavirus (hr-HPV) positive patients. First, high-throughput quantitative methylation-specific PCRs (QMSP) on a novel OpenArray TM platform, representing 424 primers of 213 cancer specific methylated genes, were performed on frozen tissue samples from 84 cervical cancer patients and 106 normal cervices. Second, the top 20 discriminating methylation markers were validated by LightCyclerV R MSP on frozen tissue from 27 cervical cancer patients and 20 normal cervices and ROCs and test characteristics were assessed. Three new methylation markers were identified (JAM3, EPB41L3 and TERT), which were subsequently combined with C13ORF18 in our four-gene methylation panel. In a third step, our methylation panel detected in cervical scrapings 94% (70/74) of cervical cancers, while in a fourth step 82% (32/39) cervical intraepithelial neoplasia grade 3 or higher (CIN31) and 65% (44/68) CIN21 were detected, with 21% positive cases for CIN1 (16/75). Finally, hypothetical scenario analysis showed that primary hr-HPV testing combined with our four-gene methylation panel as a triage test resulted in a higher identification of CIN3 and cervical cancers and a higher percentage of correct referrals compared to hr-HPV testing in combination with conventional cytology. In conclusion, our four-gene methylation panel might provide an alternative triage test after primary hr-HPV testing.Infection with high-risk human papillomavirus (hr-HPV) is causally linked to cervical carcinogenesis. 1 Cervical cancer incidence is reduced by cytologic screening, although cytologic morphologic assessment of cervical scrapings is not ideal, because its sensitivity is only $ 55% for CIN2þ. 2 Recently, preventive vaccines against hr-HPV-16 and hr-HPV-18 have been introduced in the Western world, which will reduce the incidence of cervical neoplasia significantly. However, these vaccines do not cover 100% of cervical cancers, while it will take over decades before HPV vaccination affects cervical neoplasia incidence. Therefore, screening needs to be continued. Simultaneously, efficiency of cytologic screening in population-based screening programs will be reduced by vaccination, due to a gradual decline of cervical neoplasia prevalence. Hr-HPV testing of cervical scrapings has been shown to improve sensitivity of cervical screening, 3,4 but is also associated with low specificity, especially in a young screening population. 5 For that reason, other markers are needed especially to improve the positive predictive value for population-based screening of cervical neoplasia.Promoter methylation of tumor suppressor genes has been reported to be an early event in carcinogenesis. 6 Gene pro...

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The NF-jB2/p100 and bcl-3 genes are involved in chromosomal translocations described in chronic lymphocytic leukemias (CLL) and non-Hodgkin's lymphomas, and nuclear factor kappaB (NF-jB) protects cancer cells against apoptosis. Therefore, we investigated whether this transcription factor could modulate the expression of the Bcl-2 antiapoptotic protein. Bcl-2 promoter analysis showed multiple putative NF-jB binding sites. Transfection assays of bcl-2 promoter constructs in HCT116 cells showed that NF-jB can indeed transactivate bcl-2. We identified a jB site located at position À180 that can only be bound and transactivated by p50 or p52 homodimers. As p50 and p52 homodimers are devoid of any transactivating domains, we showed that they can transactivate the bcl-2 promoter through association with Bcl-3. We also observed that stable overexpression of p100 and its processed product p52 can induce endogenous Bcl-2 expression in MCF7AZ breast cancer cells. Finally, we demonstrated that, in breast cancer and leukemic cells (CLL), high NF-jB2/p100 expression was associated with high Bcl-2 expression. Our data suggest that Bcl-2 could be an in vivo target gene for NF-jB2/p100.