Hypermethylation of tumor suppressor genes involved in critical regulatory pathways for developing a blood-based test in breast cancer.

BACKGROUND: Aberrant DNA methylation patterns might be used as a biomarker for diagnosis and management of cancer patients. METHODS AND FINDINGS: To achieve a gene panel for developing a breast cancer blood-based test we quantitatively assessed the DNA methylation proportion of 248 CpG sites per sample (total of 31,248 sites in all analyzed samples) on 10 candidate genes (APC, BIN1, BMP6, BRCA1, CST6, ESR-b, GSTP1, P16, P21 and TIMP3). The number of 126 samples consisting of two different cohorts was used (first cohort: plasma samples from breast cancer patients and normal controls; second cohort: triple matched samples including cancerous tissue, matched normal tissue and serum samples). In the first cohort, circulating cell free methylated DNA of the 8 tumor suppressor genes (TSGs) was significantly higher in patients with breast cancer compared to normal controls (P<0.01). In the second cohort containing triple matched samples, seven genes showed concordant hypermethylated profile in tumor tissue and serum samples compared to normal tissue (P<0.05). Using eight genes as a panel to develop a blood-based test for breast cancer, a sensitivity and specificity of more than 90% could be achieved in distinguishing between tumor and normal samples. CONCLUSIONS: Our study suggests that the selected TSG panel combined with the high-throughput technology might be a useful tool to develop epigenetic based predictive and prognostic biomarker for breast cancer relying on pathologic methylation changes in tumor tissue, as well as in circulation.

Levels of plasma circulating cell free nuclear and mitochondrial DNA as potential biomarkers for breast tumors.

BACKGROUND: With the aim to simplify cancer management, cancer research lately dedicated itself more and more to discover and develop non-invasive biomarkers. In this connection, circulating cell-free DNA (ccf DNA) seems to be a promising candidate. Altered levels of ccf nuclear DNA (nDNA) and mitochondrial DNA (mtDNA) have been found in several cancer types and might have a diagnostic value. METHODS: Using multiplex real-time PCR we investigated the levels of ccf nDNA and mtDNA in plasma samples from patients with malignant and benign breast tumors, and from healthy controls. To evaluate the applicability of plasma ccf nDNA and mtDNA as a biomarker for distinguishing between the three study-groups we performed ROC (Receiver Operating Characteristic) curve analysis. We also compared the levels of both species in the cancer group with clinicopathological parameters. RESULTS: While the levels of ccf nDNA in the cancer group were significantly higher in comparison with the benign tumor group (P < 0.001) and the healthy control group (P < 0.001), the level of ccf mtDNA was found to be significantly lower in the two tumor-groups (benign: P < 0.001; malignant: P = 0.022). The level of ccf nDNA was also associated with tumor-size (<2 cm vs. >2 cm<5 cm; 2250 vs. 6658; Mann-Whitney-U-Test: P = 0.034). Using ROC curve analysis, we were able to distinguish between the breast cancer cases and the healthy controls using ccf nDNA as marker (cut-off: 1866 GE/ml; sensitivity: 81%; specificity: 69%; P < 0.001) and between the tumor group and the healthy controls using ccf mtDNA as marker (cut-off: 463282 GE/ml; sensitivity: 53%; specificity: 87%; P < 0.001). CONCLUSION: Our data suggests that nuclear and mitochondrial ccf DNA have potential as biomarkers in breast tumor management. However, ccf nDNA shows greater promise regarding sensitivity and specificity.

Comparison of activin A and cell-free fetal DNA levels in maternal plasma from patients at high risk for preeclampsia.

OBJECTIVES: We examined the concentration of activin A in a prospective manner before the clinical manifestation of preeclampsia and compared the data with those of cell-free fetal DNA in the maternal plasma. METHODS: The levels of activin A were analysed by enzyme-linked immunosorbent assay (ELISA) for pregnant women: (1) with preeclampsia (n = 34) in the third-trimester and normal controls (n = 44); and (2) at-risk of preeclampsia in the second-trimester (n = 15) as indicated by uterine artery Doppler and normal controls (n = 68). Correlation between activin A level and cell-free fetal DNA level was examined using the Spearman rank test. RESULTS: The level of plasma activin A was significantly higher in the preeclamptic samples (12.056 vs 7.068 ng/mL, p = 0.000). The increase in the activin A concentration was observed prior to the onset of preeclampsia (3.483 vs 1.324 ng/mL, p = 0.000). This increase in activin A correlated significantly with the increased level of cell-free fetal DNA, in the maternal circulation prior to the onset of preeclampsia (r = 0.977, p = 0.000). CONCLUSION: Our data suggest that circulatory activin A could be an independent biomarker for the early identification and monitoring of preeclampsia.