In the current study we demonstrated a significant association between cell-free tumor DNA in blood and relapse of breast cancer patients after a follow-up time of 6 to 10 years. Furthermore, the detection of cell-free tumor DNA was not correlated to the detection of DTC in BM as an established indicator of MRD.
We detected markedly higher DNA levels in the blood of patients with lobular breast carcinomas than in patients with ductal tumors, whereas, inversely, the LOH incidence in blood of patients with ductal breast cancer was higher than in patients with lobular cancer. These observations are not easy to explain by an increased turn over of tumor cells because controversial data on the apoptotic and LOH index of the different histological cancer types have been published [19–21]. Furthermore, according to the inadequate number of patients with lobular cancer these data could also not be performed in a multivariate analysis.
Concordant LOH profiles in autologous tumor tissues and blood were only found in 3 of 22 patients where both types of samples were available, whereas discordant LOH profiles were seen in all other cases. This surprising finding may be due to the known multifocal heterogeneity of breast carcinomas , the local necrosis of the areas of the primary tumor, the potential contribution of tumor DNA derived from micrometastatic cells to the pool of blood DNA  and the masking of LOH caused by the dilution of tumor-derived DNA by normal DNA [15, 23].
Nevertheless, we discovered significant correlations between the presence of LOH in blood and the clinical parameters of our patients. The key finding was the association of LOH at the microsatellite markers D3S1255 and D9S171 with the relapse of the patients. In the literature there is little information on the marker D3S1255 mapping to the chromosomal locus 3p24.2-25. However, it has been suggested for another tumor entity that replication errors on 3p is an early event in tumor development and that a tumor suppressor gene, which is involved in the progression of esophageal squamous cell carcinoma, may exist near the 3p25 locus . Moreover, the well-known gene product CDKN2 (cyclin-dependent kinase inhibitor) is located in the vicinity of the marker D9S171. CDKN2 is a negative regulator of the cell cycle, and the loss of this gene may, consequently, promote cell proliferation, which may also explain its role in the pathogenesis of sporadic breast cancer . Besides the association of LOH at this locus with the relapse of the breast cancer patients, we found that the marker D9S171 was only affected by LOH in patients with higher tumor stages of pT2-4, indicating additionally the relationship between tumor load and the presence of such DNA in serum.
Using a well-established antibody for the detection of DTC , we found DTC in BM of nearly 40% of the patients, which is comparable with other published studies demonstrating the prognostic impact of DTC in the BM of primary breast cancer patients [1, 27–29]. In our study, the presence of DTC in BM only correlated with distant metastases but with no other clinicopathologic data. Moreover, we found no relationship between BM status and LOH on cell-free DNA in blood. This finding suggests that the presence of DTC in BM of breast cancer patients may not significantly contribute to the release of DNA into blood.
In contrast to DTC, the half life of circulating tumor cells (CTC) seems to be short (1 to 24 hours) and apoptotic cells significantly contribute to the circulating tumor cell fraction in breast cancer patients . Genomic analyses at the single-cell level have shown that DTC and CTC frequently display very heterogeneous tumor-specific aberrations, particular in patients with early-stage cancer without overt metastases [31, 32]. Furthermore, genomic and phenotypic differences between DTC, CTC and the primary tumor have been documented in breast cancer [33, 34]. As a result, it has been suggested that tumor cells disseminate in an early genomic state and that they acquire genomic alterations after dissemination independent form the primary tumor . Thus, it can be speculated that part of the DNA found in blood is derived from CTC which could also explain the discrepancies in LOH patterns observed between blood and autologous tumor tissues. We did not perform CTC analysis in the current study because no standardized method for CTC screening had been established when samples were taken 6 to 10 years ago. Future genomic analyses of CTC and DTC together with the assessment of cell-free DNA will shed more light on the origin of cell-free DNA in cancer patients.