Abstract (eng)
Changes in DNA-methylation patterns are an early event in cancer development. Many promoter regions of tumor suppressor genes or oncogenes change their methylation status due to growth advantages of the cancer cell. During this study we investigated detection of these epigenetic aberrations in cell free DNA derived from serum of breast cancer patients, for elucidation of potential biomarkers for minimal invasive diagnostics.
Therefore several DNA isolation approaches and genome wide amplification methods for enrichment of methylated DNA were tested. Applying these optimized methods serum-DNA was isolated form sera of A) breast cancer patients with malign neoplasm (n=40), B) sera of patients with a non-invasive cancer phenotype (n= 18) and C) healthy normal controls (n=24). The DNA concentrations in serum of patients with a metastasizing tumor (45,64 ng/ml +/- 32,31ng) was significantly increased when compared to serum-concentrations of normal controls (10,58ng/ml +/- 9,71ng), (p = 0.0013, Wilcoxon-test).
The methylated fraction of the sample DNA was genome wide enriched, using a restriction enzyme based approach combined with a subsequent “Rolling Circle Amplification” (RCA). After successful sample preparation of 72 out of 82 DNA samples, a biomarker screening to distinguish between the patient study groups was performed. Therefore 360 selected DNA sequences were amplified within a multiplex-PCR approach and amplicons detected upon hybridization onto a target micro-array. Micro-array analyses enabled identification of marker-candidates for classification of study groups. However upon critical examination of our experimental setup, we can not exclude that some experimental bias contributes to this discrimination.
In this work a protocol was established which enables a genome wide biomarker screening for elucidation of DNA methylation markers for minimal invasive diagnostic testing using spurious amounts of cell free serum DNA.