2.1. Specificity and Sensitivity of CDH1 and CDH13 DNA-Methylation in Serum Samples
To determine whether the DNA-methylation status of CDH1 or CDH13 in serum samples has a diagnostic value for cervical cancer, we used MethyLight analysis to investigate the frequency of DNA-methylation of CDH1 and CDH13 in serum samples from 40 patients with non-malignant diseases and 49 cervical cancer patients. In non-malignant serum samples, aberrant DNA-methylation for CDH1 was present in 10 of 40 serum samples (75% specificity). Among the patients with invasive cervical cancer, the DNA-methylation frequency for CDH1 was 55% (27 of 49; 55% sensitivity). For CDH13, we identified in non-malignant serum samples aberrant DNA-methylation in two of 40 serum samples (95% specificity), but in only five of 49 serum samples from patients with invasive cervical cancer (sensitivity 10%).
A statistically significant higher frequency of DNA-methylation for CDH1, but not for CDH13, was observed in cervical cancer patients in comparison to patients with benign diseases (p = 0.004).
2.3. Comparison of MethyLight PCR and DHPLC-PCR
Furthermore, we compared the CDH1 results of the MethyLight assay with the results of a DHPLC PCR in all 89 serum samples. We found only a modest correlation between these data (r = 0.496; p < 0.001). Only 15 of all 37 (41%) MethyLight positive samples could also be detected by means of DHPLC PCR, whereas 15 of the 16 (94%) DHPLC positive samples were detected by MethyLight PCR.
Moreover, the DHPLC method showed a statistically significant higher frequency of DNA-methylation for CDH1 in cervical cancer patients than in patients with benign diseases (p = 0.020). Among the patients with invasive cervical cancer, the DNA-methylation frequency for CDH1 was only about 27% (13 of 49). In non-malignant serum samples, we detected aberrant DNA-methylation in three of 40 serum samples (7.5%). Consequently, we identified a specificity of 92.5% and a sensitivity of only 27% for the CDH1 DHPLC PCR analysis. With this method we were not able to identify a statistically significant difference between cervical cancer patients and patients with benign diseases.
Cervical cancer is a leading cause of cancer-related death in women. Several clinical and histopathological characteristics, i.e.
, tumor stage, lymph node status, and vascular invasion, have been shown to be prognostic factors for recurrent disease [16
]. New objective diagnostic, prognostic and predictive biomarkers for cervical cancer are needed.
Recently we identified CDH1
DNA-methylation in serum samples taken at the time of diagnosis as an independent prognostic marker in cervical cancer patients with no concurrent chemo- or radiation therapy [15
In the present study, the methylation status of CDH1
genes in serum samples from 49 cervical cancer patients who were treated according to the guidelines and 40 patients with diseases other than cancer was reevaluated using MethyLight assay technology. The DNA-methylation frequency for CDH1
was 55% in the serum samples from patients with invasive cervical cancer. In our previous study, we identified a frequency of 42% in serum [15
]. Other groups describe frequencies of about 51.1–80.5% in cervical cancer tissue samples but not in serum samples. [18
]. For CDH13
methylation, a frequency of only 10% was observed in serum samples from cervical cancer patients. Distribution of CDH1
but not CDH13
methylation within the cervical cancer and non-cancer group showed statistically significant differences. For the detection of cervical cancer based on the CDH1
methylation analysis in serum samples, however, the specificity and sensitivity values of 75% and 55% are not convincing. Therefore the serological detection of CDH1
DNA-methylation in serum does not appear to be a suitable method for cervical cancer detection. There is currently no methylation marker that can be readily translated for use in cervical cancer screening or triage settings [20
To determine whether the methylation status of CDH1
in serum samples has also a prognostic value for cervical cancer, we compared serum DNA-methylation of these genes with the overall and relapse-free survival of patients. We found that there was a significant difference in the overall and the relapse-free survival for patients with methylated CDH1
DNA in their pre-treatment serum samples. These findings are in accordance with our previously published study where only patients without concurrent chemo- or radiation therapies were analyzed [15
]. In that study, we observed a higher CDH1
DNA-methylation frequency in serum samples of patients with FIGO Stage III and no association with tumor grade. In the present study, we found only a non-statistically significant trend of a higher CDH1
DNA-methylation frequency in serum samples of patients with FIGO IV and again no association with tumor grade. The low number of patients in the present study may explain the slight difference compared to the previous study. In our present study the combination of CDH1
DNA-methylation data did not improve the survival results. However, further studies with larger numbers of patients are required to confirm our findings.
Finally, we compared MethyLight technology and DHPLC-PCR for CDH1 DNA-methylation analysis. Using DHPLC-PCR, we detected only 41% of all MethyLight positive samples, but the majority (94%) of the DHPLC positive samples was also detected by MethyLight PCR.
The comparison of methods showed a higher detection rate by MethyLight PCR. Using DHPLC-PCR for CDH1,
it was not possible to distinguish statistically significantly between patients with cervical cancer and patients with benign diseases. The high sensitivity of MethyLight PCR technology could be a reason for the discrepancy in the methylation frequencies detected by MethyLight and DPHLC. MethyLight PCR can detect a single methylated allele in 105
unmethylated alleles [21
]. Previously, Eads et al. showed that MethyLight PCR is at least 10 times more sensitive than reported for the highly sensitive MSP technology [21
The findings described in this manuscript suggest that the more labor-intensive DHPLC-PCR is not an ideal method for CDH1 methylation analysis in serum samples.