Cord Beeger

INTRODUCTION: The origin and clinical relevance of circulating cell-free tumor DNA in the blood of cancer patients is still unclear. Here we investigated whether the detection of this DNA is related to bone marrow (BM) micrometastasis and tumor recurrence in breast cancer patients. METHODS: BM aspirates of 81 primary breast cancer patients were analyzed for the presence of disseminated tumor cells (DTC) by immunocytochemistry using the pan-cytokeratin antibody A45-B/B3. PCR-based fluorescence microsatellite analysis was performed for detection of loss of heterozygosity (LOH) at 6 polymorphic markers using cell-free serum DNA. The data were correlated with established risk factors, and patients were followed-up over 6-10 years. RESULTS: LOH was detected in 33.5% of blood samples. The occurrence of LOH at the entire microsatellite marker set correlated with histopathology (P = 0.05) and grading (P = 0.006) of the primary tumor. The genomic region characterized by marker D9S171 was only affected by LOH in patients with increased tumor stages (pT2-4, P < 0.05) and older age (> or = 55 years, P = 0.05). Kaplan-Meier analysis showed that LOH at D3S1255 (P = 0.009) and D9S171 (P = 0.001) were significantly associated with tumor relapse. In BM, DTC were detected in 39.5% of the patients, and this finding correlated with distant metastases (P < 0.05). Patients with DTC-positive BM had higher DNA yields in their blood than patients with DTC-negative BM (P < 0.05). However, no significant correlations were found between the presence of DTC in BM and the detection of marker-specific LOH on blood DNA. CONCLUSIONS: The detection of LOH on cell-free tumor DNA in blood is unrelated to BM micrometastasis and provides independent information on breast cancer progression.

BACKGROUND: Accurate identification of loss of heterozygosity (LOH) on circulating free DNA is often restricted by technical limitations such as poor quality and quantity of tumor-specific DNA and contamination by normal DNA. However, plasma DNA may harbor tumor-specific genetic alterations and could therefore be an interesting target for noninvasive examinations of tumor DNA. METHODS: By PCR-based fluorescence microsatellite analysis using 12 polymorphic markers, we investigated LOH on cell-free DNA in blood plasma from 59 patients with localized prostate cancer (PCa) and 12 with metastatic disease (MPCa). In addition, plasma DNA from 21 PCa patients was fractionated into high- and low-molecular-weight DNA by 2 different column systems. To avoid appearance of artificial allelic loss and stabilize the amplification, TMAC (tetramethylammonium chloride) was added to each PCR. RESULTS: Overall incidences of LOH at all markers analyzed were 10% in PCa and 12% in MPCa samples. Highest frequencies were found at markers D11S898 (28%) in PCa and D6S1631 (27%) in MPCa. Statistical evaluation showed significant associations between LOH and increasing Gleason scores for the marker combinations D6S1631*D8S286*D9S171 (P = 0.03) and D8S286*D9S171 (P = 0.05). Fractionation of plasma DNA resulted in a higher overall LOH frequency in the low-molecular-weight DNA fraction (23%) compared with the high-molecular-weight DNA (7%). CONCLUSIONS: LOH analysis of circulating DNA can provide tumor-specific genetic information on PCa patients. Fractionation of plasma DNA and addition of TMAC improved LOH detection and general assay performance.

INTRODUCTION: The aim of the study was to perform a comparative analysis of LOH (loss of heterozygosity) in primary tumors as well as peripheral blood and bone marrow (BM) of patients with breast cancer (BCa). METHODS: Performing PCR-based fluorescence microsatellite analysis and using a panel of seven polymorphic microsatellite markers, we compared the profiles of LOH in primary tumors, peripheral blood and BM plasma from patients with primary BCa (n = 40, stage M0) as well as tumor tissues and blood serum from metastatic BCa patients (n = 48, stage M1). During the course of systemic treatment blood samplings from 12 M0 and 16 M1 patients were at least once repeated. RESULTS: The overall incidences of LOH in tumor tissues, blood and BM samples were 27.5%, 9.0% and 5.0%, respectively. The marker D3S1255 was the only one in the panel that showed similar frequencies of LOH ranging from 19.0 to 24.5% in tumor, blood and BM samples. Both M0 blood serum and BM plasma samples displayed the same rate of 19.0%, whereas tumor and M1 serum showed a rate of 24.5% and 24.0%, respectively, at this locus. This marker also showed the highest frequency of LOH in serum and BM samples, whereas in tumor samples LOHs at the markers D13S218 (38%) and D17S855 (36%) were more frequent. Statistical analysis of the tumor samples showed that occurrence of LOH at the markers D3S1255 (P < 0.04), D9S171 (P < 0.05) and D17S855 (P < 0.03) correlated with undifferentiated nuclear grade. Additionally, significant associations of the number of LOH recorded at D17S250 with estrogen receptor (P < 0.02), progesterone receptor (P < 0.03) expression and high proliferation score (Ki-67 expression, P = 0.009) were observed. In blood serum samples a relationship between positive lymph node status and LOH at the marker D3S1255 was revealed (M0 stage, P = 0.05; M0+M1 stage, P = 0.004). CONCLUSION: Our study demonstrates heterogeneous profiles and low rates of LOH, particularly on free DNA in BM and blood samples. However, the significant associations of LOH with some risk factors and the demonstrated possibility of monitoring free DNA in patients undergoing systemic therapy suggest that LOH analysis may be developed into a useful diagnostic tool.

<b>Copyright information:</b>Taken from "A critical evaluation of loss of heterozygosity detected in tumor tissues, blood serum and bone marrow plasma from patients with breast cancer"http://breast-cancer-research.com/content/9/5/R66Breast cancer research : BCR 2007;9(5):R66-R66.Published online 3 Oct 2007PMCID:PMC2242661. The fluorescence-labeled PCR products of leukocytes and serum DNA were separated by capillary gel electrophoresis on a Genetic Analyzer and evaluated with the Gene Scan Analysis program. The abscissa indicates the length of the PCR product, while the ordinate gives information on the fluorescence intensity represented as peaks. The upper and lower diagram show the leukocyte DNA (reference) and serum DNA amplified with the primer binding at the D17S855. The PCR product of the serum DNA shows a LOH.

3611 Background and purpose of the study: Recent studies on breast cancer (BC) have demonstrated the prognostic significance of disseminated tumor cells (DTC) in the bone marrow (BM). However, not every patient with DTC in the BM will relapse. In this study, we compared the presence of DTC in BM and lymph node staging with genetic aberrations in circulating free DNA derived from blood samples of primary, non-metastatic BC patients. Furthermore, we evaluated, whether the frequency and pattern of allelic losses in the plasma DNA might be suitable for the identification of BC patients who might have a higher risk for relapse. Methods: Bilateral BM (10 ml) aspirations of 60 BC patients at the time of diagnosis were assessed for DTC by a Ficoll density gradient centrifugation and an immunocytochemical cytokeratin (CK) assay with the anti-CK antibody A45-B/B3. For LOH (loss of heterozygosity) analysis, genomic DNA was extracted from cell-free plasma samples and amplified using a panel of 7 polymorphic microsatellite markers mapping to known tumor suppressor genes. Tumor staging was performed by the pathologists. Results: LOH aberrations were found in 14/60 (23%) blood samples. Among the informative cases, the frequency of LOH was highest for the chromosomal loci 3p23 (14%). Further frequent LOHs were detected at 13q12-13 (D13S218, 9%), 9p21 (D9S171, 8%), 10q23.3 (D10S1765, 8%) and 17q21-22 (D17S250, 7%). At the markers 17q21 (D17S855, 4%) and 16q22-23 (D16S421, 3%) LOHs were rarely observed. In 8/14 LOH positive cases, we also detected DTC in the BM. In 3 of these 8 patients, lymph nodes were staged N1-2 and 5 patients were staged N0. In the other 6/14 LOH positive cases no DTC in BM samples were found and tumor spread to the lymph nodes could only be documented in one patient. In 11/46 LOH negative cases, no tumor load was found in the lymph nodes or in the BM whereas tumor spread to the lymph nodes (n=12) or to the BM (n=16) or to both compartments (n=5) was documented in 33/46 of these cases. Conclusions: Our results show that information on tumor specific genomic aberrations can be obtained by analysis of cell-free plasma DNA. Furthermore, these chromosomal loci harboring suppressor genes might be involved in tumor development. Thus, besides the prognostic value of DTC in BM samples, the detection and characterization of tumor-specific genomic aberrations in blood could be an additional tool to detect minimal residual disease in BC patients.