DAPK is one of the most frequently methylated genes

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, (+)-Apogossypol 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). We have performed our MSP analyses as real-time PCR using SybrGreen (Fig. 2). The results of the beta-actin PCR vary in each sample group because the amount of DNA for bisulfite treatment was adjusted to a constant volume of serum (500μl) rather than to equal amounts of DNA. Because we expected lower amounts of methylated DAPK fragments than beta-actin DNA in serum we used different amounts of bisulfite-treated DNA—in particular 5-fold more DNA for our DAPK MSP. However we still measured lower amounts of DAPK than beta-actin in sera from ovarian cancer and leiomyoma patients (Fig. 2). IVF patients showed slightly lower CT-values for DAPK than for beta-actin corresponding to equal amounts if considering the differences in input DNA (Fig. 2). To investigate the origin of methylated DNA in leiomyoma patients, we analyzed biopsies from 17 patients including 10 of the patients already analyzed for DAPK methylation in serum. However, only sera from 5 of these patients were evaluable as shown by beta-actin amplification (3 and 2 patients with DAPK positive and negative sera respectively). The frequency of DAPK methylation in primary tissue (35.3%) was higher than in serum (23.8%) but lower than the methylation frequency of ovarian cancer samples (50%, Table 2). These differences did not reach statistical significance as evaluated by the χ2-test (p>0.05). Both patients with DAPK-negative serum were also negative for DAPK methylation in the primary tissue. Moreover 2 of 3 patients with DAPK methylation in serum showed methylation in the analyzed tissue.