Detection, isolation, and initial characterization of disseminated tumor cells following enrichment in patients with prostate cancer.
Authors: Daniel W. Lin, Ilona Holcomb, Edward W. Arfman, Roger Coleman, Jesco Pfitzenmaier, William J. Ellis, Barbara Trask, Peter Nelson, Paul H. Lange, Robert L. Vessella
Journal: Cancer Research (2005)

419   Introduction and Objective: We previously reported on a disseminated tumor cell (DTC) enrichment protocol to detect and isolate single, viable disseminated epithelial cells, many of which expressed PSA from the blood and bone marrow (BM) of patients with prostate cancer (CaP). The objectives of this study were to (1) determine if detection of DTCs was associated with established clinicopathologic parameters using BM aspirates collected from patients at the time of radical prostatectomy (RP) for clinically localized CaP, and (2) determine the molecular phenotype and genomic karyotype of these cells in patients with localized and advanced CaP. Methods: 385 patients underwent BM aspiration from the anterior iliac crest immediately before RP. 30 normal controls and 11 patients with advanced CaP underwent BM aspiration from the posterior iliac crest. BM aspirates were processed using a para-magnetic epithelial cell enrichment protocol (CD61 and CD45 negative selection; human epithelial antigen (HEA) positive selection). Single DTCs and pools of 10-20 DTCs were obtained by micropipetting and then characterized by immunohistochemistry (IHC), quantitative RT-PCR, gene expression arrays and CGH arrays. Results: The median PSA of patients undergoing RP was 5.3 ng/ml (range 0.2-62.2) and tumor volume was 2.0 cc (range 0.1-40). Overall, DTCs were detected in 56% of RP patients, and PSA-positive DTCs were detected in 31%. DTC-like epithelial cells were isolated from 11% of controls; 90% of advanced stage patients were positive. The presumed DTCs, positive by HEA immunofluorescence, were also HEA positive by RT-PCR and cytokeratin positive by IHC. The detection of DTCs and PSA positive DTCs were directly associated with increasing pathologic stage, Gleason grade, serum PSA, and tumor volume (p=0.02-0.14 for multiple comparisons). The DTC population was heterogeneous in the expression of PSA and other prostate cancer associated markers. The intensity and frequency of HEA expression was most pronounced in the patients with advanced disease. Patients at high risk of progression or with advanced disease presented with DTCs that contained numerous chromosomal alterations. These alterations included several sites commonly lost and gained in CaP, i.e. loss of 8p, pericentromeric gain at 8c, and 8q gain. We also noted in some patients a high level amplification of Xq, which encompasses the androgen receptor. Conclusions: The majority of men undergoing RP for clinically localized CaP harbor these presumed DTCs in their BM. Furthermore, the detection rate of CaP cells in the BM of patients with advanced tumors is higher. These findings support the hypothesis that tumor cell dissemination is an early event and increases with aggressiveness of the tumor. Molecular and genotypic characterization is ongoing to clarify the observed heterogeneity among the DTCs.