Detection of Circulating Prostate-Specific Antigen-Secreting Cells in Prostate Cancer Patients (Technical Briefs‪)‬

The detection of circulating tumor cells in blood (1-4) requires highly sensitive, specific, and reproducible methods. During the last decade, immunocytochemistry (5, 6), reverse transcription-PCR (7-9), flow cytometry (10-13), and CellSearch and CellSpotter systems (14) have been assessed for the early detection of these rare circulating cells to predict tumor progression, survival in patients with metastatic cancer, and tumor dormancy (15). The enzyme-linked immunosorbent spot (ELISPOT) assay has been validated for enumeration of cells secreting immunoglobulins and antibodies (16,17), cytokines (18), and viral antigens (19). The 2-color ELISPOT assays allow enumeration of cells simultaneously secreting 2 cytokines (20, 21), IgG or IgA (22), or monoclonal immunoglobulins into the blood of patients with multiple myeloma (23) and MUC-1 /Cath-D-secreting cells in metastatic breast cancer patients (24). Here we describe a new ELISPOT assay for detection of human prostate-specific antigen (PSA)-secreting cells (SCs) in patients with prostatic carcinoma (PCa). This test was developed and optimized by use of LNCaP (ATCC; CRL-1740) and PC-3 (ATCC CRL-1435; provided by Pr. Pantel, Tumor Biology Institute, Hamburg, Germany) cell lines, which do and do not secrete PSA, respectively (25); PSA-SCs were then assessed in the blood of 114 men who had given written informed consent. The patients were divided into 4 groups: (a) 24 patients (median age, 73.5 years; range, 58-90 years) diagnosed with clinically localized PCa (LPCa), (b) 24 patients with metastatic PCa (MPCa), (c) 31 patients with benign prostatic hyperplasia (BPH; n = 27; median age, 69 years; range, 52-82 years) or acute prostatitis (AP; n = 4; median age, 60 years; range, 50-63 years), and (d) 35 patients (median age, 67 years; range, 22-96 years) with nonprostatic disease (NPD) and 8 healthy controls (median age, 67 years; range, 49-81 years) with serum PSA 4 txg/L. Among the patients with LPCa,12 (patients 1-12; median age, 74 years; range, 65-90 years) were studied before treatment and 12 others (patients 13-24; median age, 74 years; range, 58-86 years) were studied after treatment by radical prostatectomy (RP; n = 4; 4-6 weeks after RP), transurethral resection of the prostate (n = 5), radiotherapy (n = 2), or hormone therapy (n = 1). The characteristics of these groups are given in Table 1 of the Data Supplement that accompanies the online version of this Technical Brief at http://www.clinchem.org/content/vol51/issue8/. Among the patients with MPCa, 8 patients were studied at the diagnosis of PCa (median age, 75.5 years; range, 66-88 years) and 16 patients had metastatic localizations 2-12 years after RP (median age, 66.5 years; range, 55-81 years). Peripheral blood mononuclear cells (PBMCs), including cancer cells, were isolated by lysis of erythrocytes in blood samples collected in EDTA tubes (5-28 mL). Prostate-derived cells were enriched by the depletion of blood-derived cells by use of an anti-CD45 monoclonal antibody (mAb) together with a magnetic separation procedure (Dynal), and PSA was measured by a PSA immunometric assay (TRACE technology; B.R.A.H.M.S.).