Bone is the most frequent site of metastasis in prostate cancer and patients with bone metastases are deemed incurable. Targeting prostate cancer cells that disseminated to the bone marrow before surgery and before metastatic outgrowth may therefore prevent lethal metastasis. This prompted us to directly analyze the transcriptome of disseminated cancer cells (DCC) isolated from patients with nonmetastatic (UICC stage M0) prostate cancer. We screened 105 bone marrow samples of patients with M0-stage prostate cancer and 18 bone marrow samples of patients without malignancy for the presence of EpCAM þ single cells.

The hematopoietic growth factors granulocyte- and granulocyte-macrophage colony stimulating factor (G-CSF and GM-CSF) are nowadays widely used in routine cancer therapies as potent factors to control radiation and chemotherapy induced neutropenia, a side effect that frequently endangers the success of tumor therapies. However, there is little information about the role of G-CSF and GM-CSF for tumor growth or progression. We were interested in the expression and potential role of both factors in human meningiomas, tumors of arachnoidal origin that account for about 20% of all primary intracranial tumors. Therefore, we analyzed immunohistochemically the protein expression of G-CSF, GM-CSF and their respective receptors in 30 meningioma tissues of different malignancy and histopathological type. Both factors and receptors were not expressed in the corresponding normal tissue. In contrast, G-CSF, GM-CSF and their receptors were expressed to a varying degree in human meningiomas. Increasing expression of both factors and receptors correlated significantly with enhanced proliferation in the tumor and thus with higher malignancy. In addition, a strong perivascular expression of G-CSF was associated with a highly vascularized tumor type. Thus, expression of both G-CSF and GM-CSF is associated with the expression of proliferation vascularization, two markers of an increasingly malignant tumor phenotype, suggesting a contribution of both factors to tumor progression.

Carcinoma of the prostate is the most frequently diagnosed cancer and the second leading cause of cancer deaths in males. While it can be detected early, its progression is variable and patients that will benefit from aggressive therapy are hard to identify, leading to considerable overtreatment. Nevertheless, a fraction of patients will develop metastasis years after surgery and finally succumb to the disease. The detection of early disseminated cancer cells (DCCs) in bone marrow (BM) of carcinoma patients without distant metastasis is associated with poor prognosis. DCCs comprise precursor cells of later arising metastasis, and they are direct targets for adjuvant therapies, aiming to delay or prevent outgrowth of macrometastasis. DCCs are extremely rare and can be detected in BM by using antibodies against epithelial markers, such as cytokeratins (CKs) and epithelial cell adhesion molecule (EpCAM). We have previously observed that CK+ DCCs in BM detected in samples taken before surgery are associated with poor survival of prostate cancer patients. In contrast, we found that the detection of persisting CK+ cells in BM samples up to 8 years after surgery had no impact on the survival of prostate cancer patients, indicating that prostate cancer can become a chronic, non-progressive disease. This result prompted us to search for cells that persist after surgery and are associated with metastatic progression. We therefore tested the EpCAM in a cohort of 220 prostate cancer patients. We directly compared the prognostic impact of CK+ and EpCAM+ DCCs detected in BM samples taken months to years after surgery. Strikingly, while CK+ cells were, as previously shown, not informative about progression, the detection of EpCAM+ DCCs after surgery was associated with poor survival. Furthermore, double staining against both epithelial markers identified different subsets of DCCs. The analysis of single DCCs from BM by single cell comparative genomic hybridization (SCOMP) demonstrated that both CK+ and EpCAM+ cells are tumor cells. Interestingly, EpCAM+ cells harbored significantly more genomic aberrations than CK+ cells and are defined by characteristic changes. Since staining for EpCAM enables isolation of single cell's mRNA, we have isolated EpCAM+ cells from an additional cohort of 108 patients and from 8 healthy donors. Total genomic DNA and mRNA from these single cells were subjected to combined analysis of genome and transcriptome. PCR analysis of epithalial markers confirmed the existence of different subpopulations of EpCAM+ cells. Furthermore, using microarray analysis, we have obtained gene expression signatures of different subpopulations of DCCs. Finally, using prostate epithelial cell lines, we have investigated the functional differences of different subpopulations of EpCAM+ cells. Our results indicate an unexpected diversity of early-disseminated cancer cells displaying different phenotypes and genotypes and divergent metastatic potential. Gene expression analysis revealed great heterogeneity of the expression of analyzed markers within cells isolated from patients' BM samples. This suggests that different subpopulations of EpCAM+ DCCs may exist in diverse functional states, and may have different functional relevance. Citation Format: Miodrag Gužvić, Bernhard Polzer, Roman Ganzer, Dorothea Weckermann, Bernhard Braun, Inka Appel, Matthias Maneck, Wolfgang Wieland, Christoph A. Klein. Identification and molecular characterization of single disseminated metastasis progenitor cells in prostate cancer: The significance of EpCAM-positive cells [abstract]. In: Proceedings of the AACR Special Conference on Advances in Prostate Cancer Research; 2012 Feb 6-9; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2012;72(4 Suppl):Abstract nr A63.