Purpose: To isolate prostate epithelial cells from the peripheral blood and bone marrow, and compare prostate-specific antigen (PSA) reverse transcriptase polymerase chain reaction (RT-PCR) performed on unenriched or epithelial enriched peripheral blood and bone marrow samples.Patients and methods: Peripheral blood samples from 371 patients with prostate cancer and 141 controls, and bone marrow samples from 292 patients with prostate cancer and 43 controls were obtained. One aliquot was assessed with PSA RT-PCR. Another was enriched for epithelial cells with paramagnetic immune microbeads and assessed for: (1) PSA immunohistochemistry, (2) PSA RT-PCR, and (3) immunofluorescent detection of epithelial cells.Results: In the bone marrow (P < 0.01), but not the peripheral blood (P = 0.62), we observed significantly higher detection rates of disseminated PSA expressing epithelial cells after enrichment. The presence of epithelial cells with or without evidence of PSA production was uncommon among controls both in peripheral blood (1% and 0%) and bone marrow (11% and 0%). In patients with active prostate cancer, 46% to 74% had epithelial cells in peripheral blood, and 20% to 64% had PSA expressing epithelial cells. In bone marrow, 55% to 92% had epithelial cells, and 43% to 83% had PSA expressing epithelial cells. Particularly in bone marrow, circulating cells were frequently detected in men without evidence of disease after prostatectomy. With limited follow-up, the detection of epithelial cells or PSA expressing epithelial cells in peripheral blood or bone marrow before radical prostatectomy does not define a population of patients that will have biochemical failure.Conclusions: Immunomagnetic enrichment frequently detects epithelial, presumably malignant, cells in the peripheral blood and, especially, the bone marrow of patients with prostate cancer. Viable cells can be acquired for gene expression and phenotyping studies. (C) 2007 Elsevier Inc. All rights reserved.

OBJECTIVETo analyse telomerase activity in disseminated prostate cancer cells isolated from bone marrow aspirates taken from men with localized prostate cancer before radical prostatectomy (RP).PATIENTS AND METHODSDisseminated epithelial prostate cancer cells were isolated from bone marrow aspirates from 69 men with localized prostate cancer before RP, by magnetic column‐chromatography enrichment, followed by isolation of fluorescently labelled epithelial cells by micropipetting. We used pools of 10 non‐epithelial bone marrow cells after tumour cell enrichment as control samples. These pure cell pools were tested for the presence of telomerase activity.RESULTSIn all, 49 of the patient samples contained disseminated prostate cancer cells. Homogeneous pools of 10 cells were obtained from 35 of these; 49% of the 35 specimens showed telomerase activity, whereas all five control samples did not. Telomerase activity in the 35 samples was not significantly associated with Gleason score, preoperative prostate‐specific antigen level, tumour stage, or surgical margin status. Follow‐up is continuing to assess an association with disease recurrence.CONCLUSIONThis work shows the feasibility of isolating disseminated cancer cells for analysing individual or pooled cells. Compared to tissue staining, where telomerase is detected in 80–90% of samples, we found lower rates of telomerase activity in the disseminated tumour cells (49%). Telomerase‐negative cells might provide information about cell dormancy, as telomerase is a marker of cell proliferation in immortal and cancer cells. Telomerase‐positive cells might predict early disease recurrence, but a longer follow‐up is needed to test this possibility.

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.

You have accessJournal of UrologyDiscussed Poster, Monday, May 10, 2004, 8:00am - 12:00 pm1 Apr 2004837: Detection and Isolation of PSA Positive Epithelial Cells by Enrichment: Comparison to Standard PSA RT-PCR, Clinical Relevance, and Initial Characterization in Prostate Cancer Patients Daniel W. Lin, Jesco Pfitzenmaier, William J. Ellis, Edward W. Arfman, Jeffrey R. Klein, Paul H. Lange, and Robert L. Vessella Daniel W. LinDaniel W. Lin More articles by this author , Jesco PfitzenmaierJesco Pfitzenmaier More articles by this author , William J. EllisWilliam J. Ellis More articles by this author , Edward W. ArfmanEdward W. Arfman More articles by this author , Jeffrey R. KleinJeffrey R. Klein More articles by this author , Paul H. LangePaul H. Lange More articles by this author , and Robert L. VessellaRobert L. Vessella More articles by this author View All Author Informationhttps://doi.org/10.1016/S0022-5347(18)38086-8AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail "837: Detection and Isolation of PSA Positive Epithelial Cells by Enrichment: Comparison to Standard PSA RT-PCR, Clinical Relevance, and Initial Characterization in Prostate Cancer Patients." The Journal of Urology, 171(4S), p. 221 © 2016 by American Urological AssociationFiguresReferencesRelatedDetails Volume 171Issue 4SApril 2004Page: 221 Advertisement Copyright & Permissions© 2016 by American Urological AssociationMetricsAuthor Information Daniel W. Lin More articles by this author Jesco Pfitzenmaier More articles by this author William J. Ellis More articles by this author Edward W. Arfman More articles by this author Jeffrey R. Klein More articles by this author Paul H. Lange More articles by this author Robert L. Vessella More articles by this author Expand All Advertisement PDF DownloadLoading ...

Objectives To detect and isolate disseminated prostate cancer cells because significant effort has been directed toward defining the characteristics of the primary tumor that predict progression, but little progress has been made on evaluating the disseminated prostate cancer cell. Prostate-specific antigen (PSA) reverse transcriptase-polymerase chain reaction results in the bone marrow (BM) and peripheral blood (PB) of men with prostate cancer suggest many have disseminated cancer cells. Methods Disseminated epithelial cells were isolated from the BM and PB using Miltenyi antibody-coated paramagnetic microparticle technology. In the two-step selection process, anti-CD45 and anti-CD61 were used for negative selection, and anti-human epithelial antigen was used for positive selection. Cells were then stained for Ber-EP4 (a distinct epitope of the human epithelial antigen) and PSA. PSA reverse transcriptase-polymerase chain reaction was performed on an enriched aliquot. Results The normal controls were negative. Before prostatectomy, PSA-expressing epithelial cells were detected in 54% of BM and 24% of PB samples. At a median of 4 months after prostatectomy, PSA-expressing cells were detected in 33% of BM and 9% of PB specimens from men without evidence of disease. In men more than 5 years after prostatectomy, PSA-expressing cells were detected in the BM of 4 (29%) of 14, 2 of whom subsequently developed evidence of disease recurrence. Conclusions The findings suggest that dissemination of cells is an early event in prostate cancer that is insufficient for the development of metastases. Isolation will allow interrogation of the phenotype and genotype of the cells.

Monoclonal antibodies with high specificity for prostate tissue are of interest for prostate cancer research and treatment. Reactivity and specificity of a new murine monoclonal antibody, 107-1A4, was assessed by immunohistochemistry, ELISA and indirect immunofluorescence (IDIF). 107-1A4 stained all normal and malignant prostate tissue specimens while reactivity to non-prostate tissue was limited to the tubules of the normal kidney and renal cell carcinoma. Twenty two human cell lines were included in the reactivity survey; only the immunogen prostate cancer line LNCaP reacted with 107-1A4. Seminal plasma proteins PSA, PAP, PSMA, and PSP-94 were determined not to be the 107-1A4 antigen.

PURPOSE:The reverse transcriptase polymerase chain reaction (RT-PCR) assay is an extremely sensitive technique of detecting cells expressing prostate specific antigen (PSA). Controversy exists regarding the ability of peripheral blood PSA RT-PCR testing to reflect pathological staging or treatment outcome. We examine the phenomenology of RT-PCR results in patients with prostate cancer, with particular emphasis on the RT-PCR test before and after radical prostatectomy, and correlations with pathological staging and treatment outcome.MATERIALS AND METHODS:Peripheral blood was obtained from a wide variety of patients with and without prostate cancer, including before and after radical prostatectomy. After ribonucleic acid isolation, complementary deoxyribonucleic acid was generated and amplified with a hot-start technique. RT-PCR results were compared with pathological stage, Gleason score, tumor volume and disease-free status. Correlations between preoperative and postoperative RT-PCR tests were also made.RESULTS:The RT-PCR test was positive in 1 of 56 controls (1.8%) without suspicion of prostate cancer. A positive test was obtained in 12 of 65 men (18.5%) with a suspicion of prostate cancer but a negative biopsy. Before radical prostatectomy a positive test was obtained in 13 of 75 men (17.3%) with pT2 disease versus 10 of 46 (21.7%) with pT3 disease. There was no significant difference in serum PSA, Gleason score or tumor volume between the men with positive or negative results. With repetitive testing an increasing percentage of men had at least 1 positive test preoperatively. With a median followup of 8 months 6 of the 7 patients in whom radical prostatectomy failed had had negative RT-PCR before treatment. Of patients with known metastatic disease or failed primary treatment a positive test was obtained in 32 to 75%. Radical prostatectomy and prostate needle biopsy appeared to have a negligible effect on RT-PCR tests immediately following these procedures. Following radical prostatectomy results were variable but many men who are RT-PCR positive preoperatively become RT-PCR negative postoperatively.CONCLUSIONS:The PSA RT-PCR test in our laboratory cannot be used preoperatively to predict pathological stage of prostate cancer or treatment failure. Most cases that are positive preoperatively become negative postoperatively. While increasing tumor burden increases the likelihood of positive tests, there appears to be significant sampling error associated with the use of this test in the peripheral blood.

OBJECTIVES:To investigate the clinical value of human glandular kallikrein (hK2) reverse transcriptase-polymerase chain reaction (RT-PCR) for detection of prostate cells in circulation and to compare the results with those obtained from prostate-specific antigen (PSA) RT-PCR. METHODS:We examined peripheral blood (PB) and bone marrow (BM) samples of 13 patients with advanced-stage prostate cancer and 63 patients with clinically localized disease for the presence of circulating prostate cells. An RT-PCR protocol with a two-step amplification cycle and hot-start conditions was used. RESULTS:The limit of detection of the PCR portion is similar for PSA and hK2 (5 to 10 copies of the plasmid containing the cDNA). The RT-PCR limit of detection is one LNCaP cell in 10(8) peripheral blood mononuclear cells (PMBC) for PSA, and one LNCaP cell in 10(7) PMBC for hK2. Of the BM samples obtained prior to radical prostatectomy, 71.4% were positive for PSA mRNA and 41.3% were positive for hK2 mRNA. In PB, the PSA positivity was 19% and hK2 positivity 12.7%. In advanced-stage patients, there were 76.9% PSA-positive samples in BM versus 38.5% hK2-positive samples; 46.2% of patients were positive in PB for PSA versus 30.8% for hK2. CONCLUSIONS:We have developed a sensitive RT-PCR protocol for detection of hK2 mRNA and evaluated the suitability of hK2 mRNA in comparison with PSA mRNA as an additional marker for detection of prostate cells in circulation. Combining results of these two tests increased the sensitivity of detection.

The possibility of improving diagnosis of micrometastases from prostate cancer by further enhancing the detection of prostate-specific antigen-producing cells in circulation is being evaluated. We have developed a reverse transcriptase-PCR protocol with the desirable characteristics of low limit of detection, high specificity, reproducibility of response, and ease of performance. Among the procedural alterations that have contributed to these improvements are longer PCR primers, a two-step amplification cycle, and hot-start PCR. We have lowered the limit of detection to one LNCaP prostate-cancer cell in 10(8) peripheral blood mononuclear cells, and samples of blood and bone marrow from healthy donors have yielded no false positives. Because PCR procedures frequently exhibit tube-to-tube variability, we have incorporated a set of internal and external controls into the protocol-a significant advance in assuring assay reliability.

While prostate‐specific antigen (PSA) is already an invaluable marker for prostate cancer, there is continuing demand for new anti‐PSA antibodies with specific characteristics, e.g., high sensitivity and specificity and equimolar binding to free PSA (f‐PSA) and the PSA‐α‐1‐antichymotrypsin complex (PSA‐ACT), as well as the ability to distinguish between these 2 immunoreactive forms of PSA. We have therefore generated and characterized 10 anti‐PSA monoclonal antibodies (MAbs). Apparent dissociation constants (Kd) of MAbs were determined by direct ELISA yielding Kd‐0.2‐164.0 nM. Western blots suggested that 3 of the MAbs (60‐1A2, 60‐8A2 and 17‐1A2) bind to linear epitopes. Sandwich assays identified 5 major antigenic regions as binding targets of the MAbs. Three combinations of MAbs recognize f‐PSA and PSA‐ACT in equimolar fashion with high sensitivity. Two of the MAb combinations are specific for f‐PSA. Physical analysis of the new antibodies has allowed us to assign the MAbs to binding classes (based on their sandwiching capabilities) and to determine accurate apparent dissociation constants. Int. J. Cancer 71: 1019‐1028, 1997. © 1997 Wiley‐Liss Inc.

Radiation dosimetry and monoclonal antibody (MAB)-targeted radiotherapy studies were performed to evaluate the feasibility of using tumor-preferential MAB as targeting agents for internal radiotherapy of renal cell carcinoma (RCC). Two human RCC xenograft lines, TK-177G and TK-82, were established in nude mice and studied using MAB A6H as a targeting agent. This MAB has previously demonstrated excellent in vivo localization to RCC xenografts. Two doses of A6H (13 to 19 micrograms) labeled with iodine 131 (110 to 130 microCi) caused the tumor to regress or arrested the tumor growth in both xenografts. Similar doses (18 to 43 micrograms; 120 microCi) of 131I-labeled control MAB AFP-22 or of unlabeled A6H did not inhibit tumor growth. While most mice in the control groups had tumors greater than 250 mg in weight by day 43, none of the tumors in mice treated with 131I-labeled A6H grew to that size during the 3-month observation period. Sequential computerized scintigraphy was used to calculate the amount of radioisotope localized in tumor versus normal mouse tissue. Therapeutic doses of 131I-labeled A6H delivered a median calculated radiation dose of 38 cGy/microCi (range, 28 to 57) injected dose to RCC xenografts, and a median of 0.9 cGy/microCi to normal mouse tissues. These findings suggest that A6H is able to target radioisotopes highly specifically to RCC and achieve a therapeutic effect in the experimental setting.

Monoclonal antibodies (MABs) reactive with human renal cell carcinoma (RCC) were generated following immunization of mice with either RCC homogenates, RCC cell lines, or fetal kidney homogenates. The characteristics of four highly reactive immunoglobulin G1 MABs, designated UMVA-RCC-A6H, UMVA-RCC-A36, UMVA-RCC-C5H and UMVA-RCC-D5D are presented. The screening process consisted of a cell binding enzyme-linked immunosorbent assay and immunohistological examination of tumor, normal, and fetal tissue sections. The MABs illustrated various degrees of antigen restriction: A6H identified an antigen common to RCC, some lung and colon carcinomas, the proximal renal tubules but no other normal tissues; A36 reacted with most human tumors, the renal tubules, and many other normal tissues; C5H reacted with nearly every human cancer but of the normal tissues, only the renal glomerulus shared this antigen; D5D was very restrictive, reacting with many although not all RCC and no other cancers or normal tissues with the exception of an occasional reactivity with a Bowman's capsule. Metastatic RCC and RCC xenografts expressed these antigens. None of the MABs participated in complement-mediated cytotoxicity. In immunoprecipitation studies with L-[methyl-3H]methionine and [3H]glucosamine-HCl metabolically labeled RCC cells, C5H was shown to be associated with an antigen of Mr 115,000.