Imke Müller

Cell-free Tumor DNA in Blood Plasma As a Marker for Circulating Tumor Cells in Prostate Cancer

Purpose: Circulating cell-free DNA in the blood of cancer patients harbors tumor-specific aberrations. Here, we investigated whether this DNA might also reflect the presence of circulating tumor cells (CTC). Experimental Design: To identify the source of cell-free DNA in blood, plasma derived from 81 patients with prostate cancer was examined for CTCs and cell-free DNA. An epithelial immunospot assay was applied for detection of CTCs, and a PCR-based fluorescence microsatellite analysis with a panel of 14 polymorphic markers was used for detection of allelic imbalances (AI). Results: The plasma DNA levels significantly correlated with the diagnosis subgroups of localized (stage M0, n = 69) and metastasized prostate cancer (stage M1, n = 12; P = 0.03) and with the tumor stage of these patients (P < 0.005). AI was found on cell-free DNA in plasma from 45.0% and 58.5% of M0 and M1 patients, respectively. Detection of CTCs showed that 71.0% or 92.0% of the M0 and M1 patients harbored 1 to 40 CTCs in their blood, respectively. The occurrence of CTCs correlated with tumor stage (P < 0.03) and increasing Gleason scores (P = 0.04). Notably, significant associations of the number of CTCs with the AI frequencies at the markers D8S137 (P = 0.03), D9S171 (P = 0.04), and D17S855 (P = 0.02) encoding the cytoskeletal protein dematin, the inhibitor of the cyclin-dependent kinase CDKN2/p16 and BRCA1, respectively, were observed. Conclusions: These findings show, for the first time, a relationship between the occurrence of CTCs and circulating tumor-associated DNA in blood, which, therefore, might become a valuable new source for monitoring metastatic progression in cancer patients.

Circulating tumour-associated plasma DNA represents an independent and informative predictor of prostate cancer

To investigate whether preoperative plasma levels of free DNA can discriminate between men with localized prostate cancer and benign prostatic hyperplasia (BPH).

Identification of Loss of Heterozygosity on Circulating Free DNA in Peripheral Blood of Prostate Cancer Patients: Potential and Technical Improvements

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.

Human Prostate Cancer in a Clinically Relevant Xenograft Mouse Model: Identification of β(1,6)-Branched Oligosaccharides as a Marker of Tumor Progression

Purpose: To establish xenograft mouse models of metastatic and nonmetastatic human prostate cancer and to apply these models to the search for aberrant glycosylation patterns associated with tumor progression in vivo and in patients. Experimental Design: Prostate cancer cells (LNCaP, PC-3, LuCaP 23.1, and DU-145) were xenografted subcutaneously into immunodeficient pfp−/−/rag2−/− mice. Tumor growth and metastasis formation were quantified and as altered glycosylation patterns have been associated with metastasis formation in several other malignancies, prostate cancer cells were profiled by a quantitative real-time PCR (qRT-PCR) glycosylation array and compared with normal human prostate cells. The activity of upregulated glycosyltransferases was analyzed by their sugar residues end products using lectin histochemistry on primary tumors and metastases in the animal experiments and on 2,085 clinical samples. Results: PC-3 cells produced the largest number of spontaneous lung metastases, followed by LNCaP and LuCaP 23.1, whereas DU-145 was nonmetastatic. qRT-PCR revealed an upregulation of β1,6-N-acetylglucosaminyltransferase-5b (Mgat5b) in all prostate cancer cell lines. Mgat5b products [β(1,6)-branched oligosaccharides] were predominantly detectable in metastatic xenografts as shown by increased binding of Phaseolus vulgaris leukoagglutinin (PHA-L). The percentage of prostate cancer patients who were PHA-L positive was 86.5. PHA-L intensity correlated with serum prostate-specific antigen and a cytoplasmic staining negatively affected disease-free survival. Conclusion: We show a novel xenograft mouse model for human prostate cancer respecting the complete metastatic cascade. Specific glycosylation patterns reveal Mgat5b products as relevant markers of both metastatic competence in mice and disease-free survival in patients. This is the first description of Mgat5b in prostate cancer indicating a significant biologic importance of β(1,6)-branched oligosaccharides for prostate cancer progression. Clin Cancer Res; 18(5); 1364–73. ©2012 AACR.

Comparison of genetic alterations detected in circulating microsatellite DNA in blood plasma samples of patients with prostate cancer and benign prostatic hyperplasia.

Abstract:  Prostate cancer is the most frequent malignant disease and the second most frequent cause of death due to cancer in men in the Western world. Since serum prostate‐specific antigen (PSA) and its subforms show poor specificity in clinical practice, a molecular marker for the detection and discrimination of prostate cancer (PCa) could be of great interest. To investigate the potential significance of genetic aberrations, such as loss of heterozygosity (LOH), in PCa we identified and characterized allelic losses in circulating tumor‐associated DNA in blood from patients with localized PCa. Genomic DNA extracted from cell‐free plasma of blood samples drawn from 65 PCa patients was analyzed using a panel of 15 polymorphic microsatellite markers mapping to known tumor‐suppressor genes. Comparative analyses were performed with a control group of 36 patients with benign prostatic hyperplasia (BPH). In the current study, we demonstrate that PCa patients had higher DNA concentrations in their blood circulation than BPH patients. In the marker panel studied, LOH was more frequently detected in PCa patients (34%) than in BPH patients (22%). The incidence of LOH in the plasma DNA of PCa patients was highest at chromosomal regions 3p24 (THRB, 22%) and 8p21 (D8S360, 22%) in comparison to the BPH control cohort, which frequently showed LOH at loci 8q21, 8p21, 9p21, and 11q22 (D8S286, D8S360, D9S1748, and D11S898, each 6%). These results indicate that microsatellite analysis using plasma DNA may be an interesting tool for molecular screening of PCa patients.

Microsatellite analysis of allelic imbalance in tumour and blood from patients with prostate cancer

To investigate whether a high frequency of allelic imbalance (AI) is associated with clinicopathological variables of patients with prostate cancer.

Aberrant Presentation of HPA-Reactive Carbohydrates Implies Selectin-Independent Metastasis Formation in Human Prostate Cancer

Purpose: To investigate the impact of prostate cancer cell surface glycosylation as part of the tumor cell–endothelial cell interaction in prostate cancer metastasis. Experimental Design: Glycosyltransferase expression was profiled in metastasis-derived prostate cancer cell lines and compared with primary epithelium. Prostate cancer cells were examined for HPA- and selectin-binding and adhesion to endothelium. Spontaneous metastasis xenograft models were established to test the lectin HPA-binding sites as a marker of metastatic competence and to evaluate E-selectin-binding sites in vivo. The importance of selectins for metastasis formation was analyzed using Sele−/−/Selp−/− mice. The clinical relevance of HPA- and E-selectin-binding sites in prostate cancer was determined. Results: Glycosyltransferases involved in the synthesis of common HPA-binding sites are downregulated in prostate cancer cells. An absence of HPA-reactive carbohydrates specifically indicates spontaneous metastatic spread of prostate cancer xenografts in vivo and a poor prognosis of patients with prostate cancer. HPA-binding sites decrease in lymph node metastases compared with corresponding primary tumors. Common selectin ligands are absent on prostate cancer cells, which do not adhere to recombinant selectins or endothelium under shear stress in vitro. Spontaneous metastasis formation is largely independent of selectins in vivo. E-selectin-binding sites are detectable in only 2% of patients with prostate cancer without prognostic significance. Conclusion: Prostate cancer is characterized by an inverse functional and prognostic importance of HPA-binding sites compared with other adenocarcinomas. Accordingly, this study surprisingly shows that the selectin–selectin ligand axis, which is essential for extravasation and thus metastasis formation in several malignancies, can be circumvented in prostate cancer. Clin Cancer Res; 20(7); 1791–802. ©2014 AACR.

The PFP/RAG2 Double-Knockout Mouse in Metastasis Research: Small-Cell Lung Cancer and Prostate Cancer

Patients with small-cell lung cancer (SCLC) and prostate cancer (PCa) as well as other solid tumors may have micro- or macro-metastatic spread at an early stage of the disease. SCLC and PCa xenograft transfer models in immunodeficient mice fail to model this metastatic spread in vivo. In both tumor types the depletion of NK cells found in immunodeficient mice results in an increased number of spontaneous metastases, mirroring the clinical situation where NK cell activity in patients is related to metastatic spread of the disease. As a result NK cell activity directly influences treatment options and mortality. Newly developed immunodeficient mouse strains lacking functional T- and B-cells (rag2 knockout) however presenting functional NK cells (perforin knockout) are superior in producing spontaneous metastasis of SCLC and PCa cells compared to the system using SCID mice.