Cell-free DNA in the circulation as a non-invasive biomarker for breast cancer

Breast cancer remains the most common cancer among women worldwide. Although standard screening methods such as clinical breast examination (CBE) and Full-Field Digital Mammography (FFDM) are non-invasive itself, their sensitivity and specificity is limited and they still require biopsy proof after suspicion. Quantitative as well as qualitative changes of circulating cell-free nuclear and mitochondrial DNA (ccf nDNA / ccf mtDNA) have been shown to have potential as biomarker for breast cancer. In the first part of this study, to evaluate the applicability of plasma ccf nDNA and mtDNA quantitative alterations as a biomarker for distinguishing between three study-groups (benign, malignant, healthy), multiplex real-time PCR and ROC (Receiver Operating Characteristic) curve analysis were performed. 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). Using ROC curve analysis, we were able to distinguish between the breast cancer cases and the healthy controls using ccf nDNA as marker (sensitivity: 81%; specificity: 69%; P < 0.001) and between the tumor group and the healthy controls using ccf mtDNA as marker (sensitivity: 53%; specificity: 87%; P < 0.001). Our data suggest that both species might have a potential as biomarkers in breast tumour management. However, ccf nDNA seems to be the stronger biomarker regarding sensitivity and specificity. The second part of this work focused on finding breast cancer specific qualitative alterations in ccf nDNA and ccf mtDNA. Therefore MALDI-TOF MS based 40-plex assay was applied to investigate the mutational status of candidate cancer genes (CAN-genes) to evaluate their value as biomarker for diagnostic/therapeutic purposes. No mutations were found in the analyzed cell lines; only one breast cancer patient was found to be heterozygous at one locus within the ZFYVE26 gene which was also confirmed by single-plex assay. Sjöblom et al. / Wood et al. already showed that the vast majority of CAN-genes are mutated at very low frequency. Due to the fact that we only found one mutation in our cohort, we therefore assume that at the selected loci, mutations might be low-frequency events and therefore, more rarely detectable. However, further evaluation of the CAN-gene mutations in larger cohorts should be the aim of further studies. Compared to nDNA, the analysis of mtDNA mutations has some advantages such as higher mtDNA copy numbers and higher mutation rate. Using Sanger sequencing we identified 43 informative mutations within the HVR1 and HRV2 regions in breast cancer tissues; however we did not detect these mutations in mtDNA of the corresponding plasma samples. This might be due to the fact that the amount of circulating mutant mtDNA is tiny in comparison to background wild-type mtDNA suggesting that more sensitive methods will be needed for this approach to be of clinical utility. Since breast cancer is frequently associated with a decrease in mtDNA content which we also demonstrated in the first part of this work, we finally focused on the molecular mechanisms that are hypothesized to underlie this decrease in mtDNA content. Therefore we investigated the the PGC-1 regulatory network. Reduced mRNA expression of PGC-1α and PGC-1β was not associated with down-regulation of target genes, such as NRF1, TFB1M and TFB2M. POLRMT was downregulated in breast cancer tissues what might be implicated in decrease in mtDNA content. PGC-1β down-regulation was neither correlated with oxidative DNA damage status nor with the expression of genes of the VHL/HIF-1/C-MYC-pathway. We suspect that PGC-1β promoter methylation might be causal for its down-regulation. Summing up, limited sensitivity and specificity can be considered as the bottleneck of using quantitative as well as qualitative alterations as marker. Overall, our data give new insight to the applicability of ccf nDNA and mtDNA as a biomarker for breast cancer and contribute to a better understanding of molecular mechanisms that are suspected to underlie quantitative alterations in breast cancer.