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  • Contrary to genetic aberrations epigenetic


    Contrary to genetic aberrations epigenetic methylation markers with broad coverage of OvCa subtypes were detected in primary tumors [21]. Ibanez de Caceres et al. [22] identified hypermethylation of BRCA1, APC, p16, p14, DAPK or RASSF1A in 50 of 50 primary tumors. Moreover the methylation pattern was identical in matched serum from 41 of these patients (82% sensitivity) and serum samples from 40 control women were negative (100% specificity). Nevertheless different aspects have to be elucidated before methylation markers for OvCa can be translated into the clinic. Results from most markers were published in single studies or exhibit a wide variation of methylation frequencies within several studies [21]. Furthermore, analyses of OvCa markers in cell-free serum DNA were restricted to cancer patients and healthy controls [22], [23]. However screening markers should also be tested in patients suffering from non-cancerous disease [24]. Therefore we have analyzed the methylation of DAPK not only in tissue and serum from ovarian cancer patients but also in patients with uterine leiomyoma. Uterine leiomyoma are common benign estrogen-dependent MLN 8237 synthesis tumors with an overall prevalence of 25% [25]. The real incidence could be even higher because only a subset of women develops clinically conspicuous symptoms and small leiomyomas are frequently not detected in the postmenopause [26].
    Materials and methods
    Results The differentiation between methylated and unmethylated DNA by MSP relies on the sequence-specific amplification of bisulfite treated DNA. Methylated cytosines are protected from bisulfite induced deamination whereas unmethylated cytosines are deaminated to uracil. Primers for methylation specific PCR should clearly distinguish between sequence deviations reflecting the methylation state of the original DNA. To evaluate the analytical specificity and sensitivity of the MSP potentially unmethylated DNA, SssI methylated DNA and a dilution series of methylated DNA (in-vitro SssI methylated HeLa DNA) within unmethylated DNA (HeLa genomic DNA) were used (Fig. 1). These experiments clearly demonstrated both the high specificity and sensitivity of our MSP protocol. All analyzed controls from healthy individuals were not detected by DAPK primers for methylated DNA. Additional experiments with several commercially available unmethylated DNAs confirmed these results (data not shown). Moreover this specificity is accompanied by a high sensitivity (Fig. 1B). Methylated DAPK sequences were detected down to a dilution of 1% M-DNA in 100ng UM-DNA prior to bisulfite treatment corresponding to 8 cell-equivalents in the PCR reaction (not assuming DNA degradation during bisulfite treatment). The beta-actin control amplification which is independent of any CpG methylation confirms the presence of equal amounts of DNA in each dilution. Therefore we conclude that this assay is able to detect specifically and sensitively methylated DAPK sequences in clinical samples. DAPK is one of the most frequently methylated genes in ovarian cancers [21]. Moreover DAPK can be hypermethylated in cancer of the bladder, lung, colorectum, cervix uteri and in lymphoma or glioma [31], [32], [33]. We have examined 32 late stage ovarian carcinomas (FIGO III/IV) of different histological subtypes (Table 1). Beta-actin could be amplified in bisulfite treated DNA of 28 carcinomas. Half of these carcinomas (n=14) also exhibited detectable methylation of DAPK (Table 2). We observed similar methylation frequencies in histological subgroups (serous, papillary and endometrioid OvCa 52%, 40% and 25%, respectively). We then analyzed serum samples of the same ovarian cancer patients (n=32, preoperative sera) and sera from patients with leiomyoma (n=30) and from patients of our reproductive medicine unit (IVF, n=20) as controls. Only sera with positive beta-actin amplification were judged as evaluable. Therefore 23, 21 and 8 samples from OvCa, leiomyoma and IVF patients, respectively, were analyzed for DAPK methylation. The observed 56% methylation frequency in ovarian cancer sera was comparable to the primary tumors (13/23 samples, Table 2). However, methylation did not correlate for tumor DNA and matched serum samples (Kappa=-0.13). Only 5 of 10 DAPK-positive tumors showed also methylated sequences in preoperative serum whereas 8 patients were positive for DAPK methylation in serum only. Moreover methylated DAPK DNA was detected in 5 of 21 leiomyoma patient sera and in 4 of 8 serum samples from women attending our IVF unit (Table 2). Thus the DAPK methylation frequency was significantly lower in leiomyoma sera in comparison to OvCa sera (p<0.05).