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Technical Briefs |
1 Laboratoire de Virologie and7
Service d’Urologie, Hôpital Lapeyronie, Montpellier, France;
2 INSERM U475, Immunopathologie des Maladies Tumorales et Autoimmunes, Montpellier, France;
3 Service d’Urologie, Clinique Beau-Soleil, Montpellier, France;
4 Laboratoire de Biologie Cellulaire and6
Departement d’Information Médicale, Hôpital Arnaud de Villeneuve, Montpellier, France;
5 INSERM U540, Endocrinologie Moléculaire et Cellulaire des Cancers, Montpellier, France;
8 UMR 2714 bioMerieux/CNRS, IFR 128 BioSciences Lyon-Gerland, Lyon, France;
aaddress correspondence to this author at: Laboratoire de Virologie, Hôpital Lapeyronie, 291 Avenue du Doyen Giraud, 34295 Montpellier, France; fax 330-467-338-334, e-mail jp-vendrell{at}chu-montpellier.fr
The detection of circulating tumor cells in blood (1)(2)(3)(4) requires highly sensitive, specific, and reproducible methods. During the last decade, immunocytochemistry (5)(6), reverse transcription-PCR (7)(8)(9), flow cytometry (10)(11)(12)(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 µg/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.).
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The PSA ELISPOT assay was performed as described recently for MUC-1/Cath-D (24). Briefly, Immobilon-P membranes were coated with mAb 11E5C6 (0.9 mg/L; bioMerieux) directed against total PSA (26), and 105 to 1 LNCaP cells and 5 x 104 to 2 x 105 of enriched CD45– cells from patients with PCa were seeded in 4 different wells. After 24–48 h of cell culture, alkaline phosphatase- or phycoerythrin-labeled anti-free PSA mAb 6C8D8 (0.3 and 0.9 mg/L, respectively; bioMerieux) was added (26). The percentages of cytokeratin-negative (CK–) and PSA-positive LNCaP and PC-3 cells were determined by flow cytometry (FC 500 apparatus; Beckman-Coulter) by intracytoplasmic staining by isothiocyanate of fluorescein-conjugated anti-human CK mAbs (DakoCytomation), phycoerythrin-conjugated anti-human PSA mAbs (bioMerieux), and the IntraPrepTM permeabilization reagents (Beckman-Coulter). LNCaP, PC-3, and CD45– cells (6 x 104) from 2 MPCa patients (patients 29 and 44) and from 2 healthy controls were seeded on glass slides and immunostained for PSA and CK.
LNCaP cells taken between doubling times 1 and 20 were incubated for 24 h on the ELISPOT plate (n = 6); a mean (SD) of 29 (4.9)% of the cells secreted PSA, whereas PC-3 cells did not. Flow cytometric analysis (n = 6) showed that 100% of the cells of the 2 lines secreted intracytoplasmic CK; 27 (2.1)% of the LNCaP cells but none of the PC-3 cells were PSA-positive. Finally, similar results were obtained with immunocytochemistry experiments (n = 6; Fig. 1 of the online Data Supplement). The threshold for detection of the PSA-secreting cells by the PSA ELISPOT assay was estimated by serial dilutions of the LNCaP cells (105 to 1 cell/well). The PSA ELISPOT assay detected cells at concentrations 4 orders of magnitude lower than did the PSA immunoassay in culture supernatants (Table 1
). To increase the sensitivity of the PSA ELISPOT assay, erythrocyte lysis and CD45+ cell depletion were performed before the remaining cells were tested for PSA secretion by dispersion of 103, 102, 10, and 1 LNCaP cells in 10 mL of control blood (Table 1
).
In patients without prostate cancer, no PSA-SCs were detected in the blood of 27 patients with BPH, 4 patients with AP (Table 2 of the online Data Supplement), and 35 with NPD (Table 3 of the online Data Supplement) or in 6 healthy controls (data not shown), although most had abnormally increased serum PSA. Among 12 PCa patients tested before treatment, 5 (42%) had detectable PSA-SCs (median, 9 cells; range, 2–172 cells), whereas for the others no PSA-SCs were detected (Table 1
of the online Data Supplement). These results could be explained by the absence of cell shedding by the primary tumor or by the random distribution of a small number of PSA-SCs (27). Serum PSA (median, 34 µg/L; range, 22–5300 µg/L) was significantly higher (P = 0.001) in patients with PSA-SCs than in those without circulating cells (median, 15 µg/L; range, 4.5–20 µg/L), but the cell numbers were not correlated with the serum PSA concentration (r = 0.31; P = 0.12). For 12 patients eligible as responders to treatments because all were asymptomatic, bone-scan negative, and without biochemical recurrence, serum PSA (median, 1.12 µg/L; range, 0–4 µg/L) was significantly lower than in patients tested before treatment (median, 19.5 µg/L; range, 4.5–5300 µg/L; P = 0.0001), and no PSA-SCs were observed. The number of patients who had PSA-SCs before treatment was significantly higher than the number of patients after efficient treatment (5 of 12 vs 0 of 12; P = 0.04). This observation suggests that elimination or therapeutic control of the prostate tumor process stopped the shedding of cancer cells into the bloodstream. Among MPCa patients, 20 (83.3%) had PSA-SCs in the blood (Table 1
of the online Data Supplement; median PSA-SCs, 21.5; range, 0–684; median serum PSA, 92.5 µg/L; range, 0.3–8400 µg/L). The percentage of MPCa patients with PSA-SCs was significantly higher than the percentage of patients with LPCa (20 of 24 vs 5 of 12; P = 0.02). For 2 MPCa patients, positive cells were observed by CK/PSA immunocytochemistry (Fig. 2 of the online Data Supplement). In contrast, no CK- or PSA-positive cells were detected in the blood of the healthy controls, suggesting that the PSA-SCs in MPCa patients probably represented cells derived from the primary tumor. The specificity and sensitivity of the PSA ELISPOT assay were 100% (95 confidence interval, 94.6%–100%) and 69.4 (54.4–84.5)%, respectively, and the positive and the negative predictive values were 100 (86.3–100)% and 85.7 (77.9–93.5)%, respectively.
The ELISPOT assay offers advantages compared with other methods: (a) It has a resolution orders of magnitude greater than that achieved by flow cytometry and PSA immunometric assays because the secreted PSA proteins are immunocaptured by the membrane in the immediate vicinity of the cells before being diluted in the culture supernatants. (b) In contrast to the real-time reverse transcription-PCR technique, the ELISPOT assay is based on the identification of only viable functional cells targeted by the proteins they secrete. (c) The anti-PSA antibodies used in the PSA ELISPOT assay have been carefully selected for their high specificity, which eliminates false positives. The PSA ELISPOT assay is, however, time-consuming because 48 h of cell-culture is necessary for immunocapture of the secreted PSA by the ELISPOT support.
The combination of the PSA ELISPOT assay with a cell-enrichment procedure and cell cryopreservation allowed the detection of as few as 1 PSA-secreting LNCaP cell disseminated in 10 mL of control blood. In addition, control PBMCs did not secrete PSA, suggesting that residual PBMCs remaining after immunomagnetic enrichment do not interfere with the PSA ELISPOT assay. Consequently, this ELISPOT assay appears to be able to detect a single PSA-SC in 20 mL of blood from patients with MPCa or LPCa. The number of circulating prostate-derived cells was not significantly correlated with the serum PSA concentration. PSA-SCs were enumerated in the majority of the PCa patients, whereas they were not detected in patients with BPH, AP, or NPD or in healthy controls, in agreement with previous reports (28). This observation strongly suggests that circulating PSA-SCs were shed only by the malignant prostate tumors. The presence of these cells was more frequent in PCa patients before rather than after treatment and in patients with MPCa rather than LPCa, suggesting that detection of these cells could be used as a marker to measure therapeutic efficacy and tumor progression in PCa patients. We recommend that PSA ELISPOT assays be performed on more patients with PSA values between 4 and 10 µg/L because the results of those experiments may be of interest for clinicians to differentiate patients with LPCa from patients with benign disease.
Acknowledgments
This work was supported by grants from the Ministère de l’Economie des Finances et de l’Industrie (MINEFI), the Association Régionale de Recherche et de Consensus en Onco-Urologie du Languedoc-Roussillon (ARCOU), and from the Centre Hospitalier Universitaire de Montpellier (France). We are grateful to F. Bel, A. Bouananni, and N. Mialanes for excellent technical assistance. We are indebted to Dr. S. L. Salhi for presubmission editorial assistance.
References
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