Detection of tyrosinase mRNA in melanoma by reverse transcription-PCR and electrochemiluminescence

¹ Biotechnology Division, Chemical Science and Technology Laboratory, National Institute of Standards and Technology, Gaithersburg, MD 20899.

² Department of Molecular Oncology, John Wayne Cancer Institute, Santa Monica, CA 90404.
^a Author for correspondence. Fax 310-330-3447; e-mail coc{at}nist.gov.

	Abstract

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Increased sensitivity and improved quantitation of analytical tests used in biotechnology and clinical chemistry are goals of many laboratories. We have used tyrosinase primers to specifically amplify by RT-PCR the tyrosinase mRNA expressed by the M12 melanoma cell line in a background of mRNA from breast cancer cells. An electrochemiluminescence detection procedure was used as a readout system for this study. Biotinylated post-PCR cDNA samples were hybridized to a tris(2,2'-bipyridine)ruthenium(II) (TBR) chelate-labeled oligonucleotide probe, and the hybrid was subsequently captured by streptavidin-coated Dynabeads®. When either the QPCR System 5000 or the Origen 1 Analyzer System were used, the luminescence emitted by the TBR-chelate of the captured specific post-PCR product was assessed. Tyrosinase-specific mRNA isolated from ~1–10 melanoma cells in a background of 10⁷ cells could be detected. We improved the sensitivity and logistics of the assay through the use of rTth for reverse transcription and amplification. Tyrosinase mRNA was detected in blood from 7 of 16 melanoma patients, whereas none of the 5 healthy donor bloods were positive (P = 0.01; Wilcoxon test).

	Introduction

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Malignant melanoma is the most rapidly increasing cancer in the United States; the incidence has increased multifold in the United States during the past 35 years (1). It has been projected that by the year 2000, ~1 in 90 Americans will develop melanoma in their lifetime (2)(3)(4). Therefore, there is much interest in developing sensitive diagnostic assays to detect this cancer. Such an assay would be used for initial diagnosis and staging to determine treatment, detection of metastases, and assessment of responsiveness to therapy. A PCR-based assay for melanoma was reported by Smith et al. (5) in 1991. These investigators successfully used oligonucleotide primers to amplify tyrosinase mRNA from the peripheral blood of patients with malignant melanoma, using a reverse transcription-PCR (RT-PCR)¹ protocol. The tyrosinase gene has been used for these studies because the enzyme is involved in the melanogenesis pathway in mammals and is synthesized primarily by melanocytes localized in the skin (6). Melanoma cells are derived from melanocytes, and both of these cell types express the tyrosinase gene. The cell specificity of tyrosinase gene expression makes it ideal for detecting metastatic melanoma cells in the blood, lymph nodes, or other organs.

Studies by Hoon and co-workers (7)(8) have demonstrated the clinical utility of using tyrosinase mRNA as a molecular marker for detection of metastatic melanoma cells in the blood and lymph nodes. The RT-PCR assay is far more sensitive for detection of melanoma cells in the blood compared with histochemical techniques. Assessment for tumor cells in the blood provides potential opportunity to monitor metastatic disease spreading, to modify clinical staging, and to monitor treatment efficacy. The RT-PCR assay using specific tumor-associated markers for detection of metastatic tumor cells in blood has been shown quite useful and practical in many studies (5)(7)(8). However, improvements are needed in the final readout of RT-PCR cDNA products. To date, most approaches described for examining PCR products of clinical specimens, such as gel electrophoresis with ethidium bromide staining, have been subjective.

An electrochemiluminescence (ECL) system (9) was used to assess the RT-PCR product of tyrosinase mRNA from melanoma cells. Briefly, the system involves a biotinylated PCR product hybridized to a tris (2,2'-bipyridine) ruthenium (II) (TBR) chelate-labeled oligonucleotide probe; the resulting hybrid is captured on magnetic beads for activation of the TBR and quantitation. The hybridization of the tyrosinase oligonucleotide probe to the PCR product confers specificity to the reaction, and detection is achieved when the TBR-labeled probe emits light (luminesces) after electrochemical oxidation.

Our results indicate that this system can detect sensitively tyrosinase mRNA expressed by melanoma cells from in vitro and in blood of melanoma patients. In the in vitro model, the assay detected tyrosinase-specific mRNA isolated from a small number of melanoma cells in a background of 10 nonmelanoma cells. Additional dilutions of the melanoma cells were detected when an additional step of nested PCR reaction was performed. We improved detection substantially through the use of rTth(TM) DNA polymerase for reverse transcription (RT) and amplification. The protocol is markedly quicker and less laborious than the gel electrophoresis followed by Southern transfer, hybridization, and autoradiographic imaging commonly used to detect specific PCR products. Although ECL can be quantitative, the tyrosinase assay that we have developed was optimized for the detection limit rather than for quantification.

	Materials and Methods2

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rna sources
M12 and M101 are established melanoma cell lines (10)(11). The human B231 cell line (MDA-MB231) was obtained from the American Type Culture Collection, Rockville, MD. Blood was obtained from healthy donors and melanoma patients. Peripheral blood lymphocytes were isolated from blood by Ficoll-Hypaque gradient centrifugation and washed several times. The M12 human melanoma cells were diluted into 10 breast cancer cells (B231 cell line). Controls included healthy donor peripheral blood lymphocytes, B231 (negative controls), and M12 and M101 (positive controls). Total RNA was extracted using the STAT-60 kit (TEL-TEST "B"). Blood cells from melanoma patients were procured as described above; the initial needle puncture containing the skin plug was discarded to prevent contamination with nondiseased skin. Blood procurement from patients was approved through the human subjects committees of the John Wayne Cancer Clinic and Saint John's Health Center. All melanoma patients had been clinically diagnosed with melanoma [American Joint Committee on Cancer (AJCC) II, III, or IV] and had given written consent for blood drawing. Patients A, B, K, and P had AJCC stage II disease; patients C, D, E, F, H, I, L, M, and O had AJCC stage III disease; and patients G, J, and N had AJCC stage IV disease. Healthy volunteer donor bloods (Q–U) were collected in the same manner as patient bloods.

oligonucleotides
Oligonucleotides used for PCR primers and ECL probes were purchased from Bioserve Biotechnologies and purified by HPLC. Nested sense and antisense primers were synthesized without biotin and then were 5' end-labeled with biotin to test the efficiencies of both sense and antisense probes in the ECL detection of tyrosinase. Probes were 5' end-labeled with TBR (Perkin–Elmer). Primer and probe sequences are shown in Fig. 1 B. Primer sequences were identical to those published by Smith et al. (5). Probes were selected from the tyrosinase sequence (12) within the nested PCR product region (Fig. 1 ) and analyzed for thermodynamic and structural variables, using the Generunner program (Hastings Software). The antisense probe covers the E2-E3 splice junction, which contains 9 nucleotides from exon 2 and 10 nucleotides from exon 3.

View larger version (24K): [in this window] [in a new window]   Figure 1. Organization of the human tyrosinase mRNA and location of RT-PCR primers, probes, and amplified regions. (A) Boxes I-V represent the five exons of the tyrosinase gene. The positions of the predicted RT-PCR products amplified by outer primers HTYR 1 and 2, HTYR 3 and 4, and HTYR 3 and 2 are shown with sizes of amplification products indicated in bp. () Position of the antisense probe; the position of the sense probe is in exon 2, 136 bp upstream from the antisense probe sequence. (B) Oligonucleotide primers and probes used for tyrosinase amplification and detection as shown in A.

amplitaq rt-pcr amplifications
The RT-PCR assays were performed using AmpliTaq® DNA polymerase (GeneAmp RNA PCR kit, Perkin–Elmer Cetus). For RT of tyrosinase mRNA, 1–2 µg of total RNA was used as template in a 20-µL reaction containing 1x PCR buffer (10 mmol/L Tris-HCl, 50 mmol/L KCl, pH 8.3), 1.0 mmol/L each dNTP, 1 unit of RNase inhibitor, 2.5 units of Moloney murine leukemia virus reverse transcriptase, and 5 mol/L outer antisense primer (Table 1 ). After incubation at 37 °C for 1 h, the enzyme was inactivated at 99 °C for 5 min, and the product was diluted to 100 µL for amplification. PCR amplification was carried out in the following: 1x PCR buffer, 200 µmol/L each dNTP, 2 mmol/L MgCl₂, 1 µmol/L outer sense and antisense primer, and 2.5 U of Taq DNA polymerase in a Perkin–Elmer 480 Thermal Cycler for 35 cycles.

For the nested PCR reactions, 10 µL of product from the first amplification was re-amplified for 35 additional cycles, using 0.2 µmol/L nested primers. Single-step PCR was performed under the same conditions, except that 0.2 µmol/L biotin-labeled nested 5' sense primer and 0.2 µmol/L outer antisense primer (unlabeled) were used for a 40-cycle amplification.

rTth RT-PCR AMPLIFICATIONS
RT and PCR reactions were performed in a single reaction tube, using rTth for both cDNA synthesis and PCR amplification at an increased temperature (GeneAmp® EZ rTth RNA PCR kit, Perkin–Elmer Cetus). Conditions were as follows: 1 µg of total RNA was amplified in a total volume of 50 µL containing 50 mmol/L bicine, 115 mmol/L KC₂H₃O₂, 80 g/L glycerol, pH 8.2, 0.45 µmol/L primers, 300 µmol/L each dNTP, 2.5 mmol/L Mn(CH₃COO)₂, and 5 U of rTth DNA polymerase. Sample evaporation was avoided by using AmpliWax® Gem 50 beads in the reactions (Perkin–Elmer). The manufacturer's recommendations for RT were followed (60 °C for 30 min). Amplification of cDNAs followed RT immediately, using three linked files: a denaturation cycle at 94 °C for 120 s; 40 amplification cycles at 94 °C for 45 s and at 60 °C for 45 s, and a final extension cycle at 60 °C for 7 min.

electrochemiluminescence detection
Biotinylated samples were analyzed using either the QPCR System 5000 (Perkin–Elmer) or the Origen 1 Analyzer System (IGEN) after hybridization and bead-capture reactions as indicated in specific experiments. For hybridization, 0.2–5 µL of PCR product was added to 40–44.8 µL of 1x PCR II Buffer (10 mmol/L Tris-HCl, pH 8.3, 50 mmol/L KCl) and 1–5 µL of TBR probe (10 µmol/L; Bioserve Biotechnologies) in a final volume of 50 µL. The mixture was heated to 95 °C for 5 min for denaturation and cooled to the optimal annealing temperature for probe hybridization (65–70 °C) for 10 min, using the Perkin–Elmer 480 Thermal Cycler. For experiments analyzed on the QPCR 5000 System, hybridized PCR cDNA products were captured on QPCR streptavidin-coated Dynabeads® (Dynal; 20–30 µg) through incubation at 55 °C or room temperature for 30 min with gentle shaking. After the DNA hybrids were captured on the beads, the total reaction volume was transferred to a separate QPCR System tube containing 400 µL of assay buffer for measurement of the electrochemiluminescent signal. For samples analyzed on the Origen Analyzer, hybridized PCR cDNA products were captured on 6.25 µg of M-280 streptavidin-coated Dynabeads by incubation at room temperature for 30 min with shaking. All other reaction conditions were the same for the two systems.

	Results

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Following the hybridization-based protocol for the QPCR 5000 system (9), cDNA was synthesized with tyrosinase-specific primers and then amplified. Primers used for amplification of the tyrosinase cDNA (5) are listed in Fig. 1B . A map of the tyrosinase gene and the locations of these primers are shown in Fig. 1A . Two oligonucleotide probes were synthesized from sequences within the tyrosinase gene segment amplified in the nested PCR reaction (Fig. 1B ). Both were tested in the QPCR protocol, and the antisense probe was found to produce higher luminescence units than the sense probe synthesized from sequences immediately adjacent to the antisense probe (Fig. 2 ). As predicted, the antisense probe hybridized to the magnetic-captured biotin-labeled PCR cDNA product and was detected, whereas the sense probe hybridized to the unlabeled product and was washed away by the assay buffer to give a background signal. Therefore, the antisense probe was used in all subsequent experiments.

View larger version (14K): [in this window] [in a new window]   Figure 2. Tyrosinase probe selection. Biotinylated PCR products (0.04, 0.2, 1.0, and 5 µL) were added to sense (dashed line) or antisense (solid line) TBR-labeled probes and hybridized. Probe concentration for hybridization was 1.0 µmol/L. Samples were applied to the QPCR System 5000, and luminescence was measured.

The optimal concentration of probe necessary to detect the tyrosinase-specific product was determined (Fig. 3 A). Antisense TBR-labeled probe (1–50 pmol) was added to 1.0 µL of a PCR product with known luminescent activity under established QPCR conditions. No concentration-dependent differences were observed in this range of probe concentrations.

View larger version (13K): [in this window] [in a new window]   Figure 3. Tyrosinase probe (A) and magnetic bead optimization (B). (A) TBR-labeled antisense probe (1.0–50.0 pmol in 5-pmol increments) was added to 1.0 µL of PCR product for hybridization (final probe concentrations per hybridization were 20 nmol/L to 1.0 µmol/L). After detection of the luminescent signal, luminosity was plotted vs amount of probe. (B) QPCR streptavidin Dynabeads (5.0–60.0 µg in 5.0-µg increments) were added to hybridization products. After QPCR detection, luminosity was plotted vs quantity of Dynabeads.

To determine the optimal concentration of magnetic beads to capture the tyrosinase-specific PCR product and probe, bead quantities were varied from 5 to 60 µg per 50-µL reaction in 5 µg increments (Fig. 3B ). The probe concentration was maintained at 200 nmol/L (10 pmol/50 µL reaction), and 1.0 µL of PCR product was used. The number of luminescent units increased as the amount of PCR product was increased from 5 µg to 15 µg, plateaued between 15 µg and 30 µg, then gradually declined. All experiments on the QPCR 5000 were subsequently performed using 30 or 20 µg of magnetic beads.

To determine the sensitivity of the method for detecting tyrosinase mRNA expression, M12 melanoma cells were diluted into 1 x 10 B231 breast cancer cells in 10-fold increments from 1 x 10 to 1 x 10 M12 cells/sample. RNA was isolated from the dilution series, and 1–2 µg was used for these experiments. RNA integrity was checked by RT and PCR amplification for 20 cycles with primers for human ß-actin; signals for products were similar for all samples tested (data not shown). The first four groups of studies (Table 1 ) performed used established methods for tyrosinase amplification that include two amplification steps with nested primers sets. The results of different experimental conditions are shown in Fig. 4 A. Specific amplification and QPCR detection assay conditions are described in Table 1 . In all studies, luminescent units above background could be detected when RNA from 10¹ M12 cells diluted in 10 B231 cells was amplified. The background was considered as the luminosity when the assay was run without tyrosinase mRNA. In two groups (2 and 4), RNA from 10 cells was detectable after amplification (Fig. 4A ). In both of these studies, the concentration of primer used for amplification was 0.2 µmol/L. Both the increase of primer concentration to 1.0 µmol/L and the decrease of primer to 0.1 µmol/L appeared to increase the detection limit of the assay. The 0.2 µmol/L primer concentration was used in all subsequent studies (Table 1 ). Varying the experimental conditions demonstrated that, in most conditions except group 3, luminosity increased as the number of M12 cells increased.

View larger version (17K): [in this window] [in a new window]   Figure 4. Comparison of nested vs single round PCR for tyrosinase mRNA detection of M12 cells diluted into 107 B231 cells. The specific QPCR conditions for each experiment are presented in Table 1 . B231 cells alone were considered as background. (A) QPCR detection of tyrosinase mRNA in different amounts of M12 cells, using nested primer pairs (groups 1–4, Table 1 ): (), group 1; (), group 2; (), group 3 (performed in triplicate); and (), group 4 (performed in duplicate). Data points are means with SD bars. (B) QPCR detection of tyrosinase mRNA in different amounts of M12 cells, using a single round of PCR amplification (groups 5–8, Table 1 ): rTth polymerase was used in groups 5 (), 7 (), and 8 (); murine leukemia virus reverse transcriptase and AmpliTaq DNA polymerase were used in group 6 (). Data points are means of duplicate experiments with SD bars.

We previously reported that the limit of detection for amplification of tyrosinase mRNA expression in melanoma cells was better when rTth DNA polymerase was used for RT and PCR amplification (13). Additional experiments were conducted to determine whether two amplification reactions were required for sensitive detection of tyrosinase mRNA expression using the QPCR 5000 system. Both AmpliTaq and rTth polymerases were used in a single round of amplification, and the concentration of RNA in the AmpliTaq amplification reaction was adjusted for a direct comparison of the two polymerases (Table 1 , Fig. 4B ). In all four groups, tyrosinase mRNA for 10 M12 cells was detected above background 0 (Fig. 4B ). In all three rTth polymerase groups (Table 1 ), luminescence was detected above background values for all dilutions of M12 cells. In two of these groups (7 and 8), the luminescence units were slightly above the B231 cell background at 1 M12 cell/10 B231 cells, and luminescent units increased rapidly when the number of cells was increased from 1 to 10 in group 5. The data shown in Fig. 4B demonstrated that using rTth polymerase in a combined RT plus PCR reaction resulted in greater detection than separate RT plus AmpliTaq polymerase at the lower numbers of cells. Only one other parameter appeared to positively influence tyrosinase mRNA detection using AmpliTaq polymerase. When the amount of RNA was doubled for a single round of amplification using RT and AmpliTaq polymerase vs nested RT and PCR, we were able to increase detection to 100 M12 cells/10 B231 cells (Fig. 4 ). Overall, rTth was generally more sensitive in detecting tyrosinase mRNA at the lower concentrations of melanoma cells. In addition, the procedure using rTth was much simpler, which in turn eliminated the chance of inducing contamination, as so often occurs with nested primer RT-PCR. Therefore, rTth polymerase was used in subsequent experiments.

We transferred the assay from the QPCR 5000 to the Origen 1 Analyzer. The systems are comparable except that the latter is easier to use. To validate the studies of the QPCR 5000 system using the Origen 1 Analyzer, RNAs isolated from the M12 dilution series were reexamined. The Origen 1 Analyzer required lower quantities of M-280 magnetic beads, thus reducing assay costs (Fig. 5 ). The optimal concentration of beads was ~0.125 g/L when 50 µL of bead solution was used for each assay sample. The luminosity decreased with higher concentrations of beads. The luminescence units detected in positive samples were considerably higher in the Origen 1 Analyzer, but the overall rate of detection was similar. The major concentrations of beads used for capture of product were compared with the number of M12 cells detected (luminosity) in Fig. 6 . One to 10 M12 cells in 10 B231 cells were easily detected with 0.125 and 0.25 g/L bead concentrations (Fig. 6 ).

View larger version (19K): [in this window] [in a new window]   Figure 5. Magnetic bead optimization for the Origen 1 Analyzer. Detection of tyrosinase mRNA of M12 cells diluted in 107 B231 cells. rTth was used for both cDNA synthesis and PCR amplification. M12 cell dilutions were 0 (), 1 (), 10 (), 102 (), and 103 (). M-280 streptavidin Dynabeads were added to hybridization products at concentrations of 1, 0.5, 0.25, 0.125, 0.1, 0.075, and 0.05 g/L (equivalent to 50 µg beads/reaction and less, respectively). These are representative experiments.

View larger version (17K): [in this window] [in a new window]   Figure 6. ECL detection of tyrosinase mRNA expression of the M12 cells. Detection of tyrosinase mRNA of M12 cells diluted in 107 B231 cells. rTth was used for both cDNA synthesis and amplification. Experiments show different concentrations of M-280 streptavidin Dynabeads: (), 0.125 g/L; (), 0.25 g/L; (), 0.5 g/L; and (), 1 g/L magnetic bead capture of tyrosinase cDNA product from different numbers of M12 cells. These are representative experiments.

To test the Origen 1 with clinical samples, we obtained blood from melanoma patients at various stages of disease. Total RNA was extracted from the blood and assessed for tyrosinase mRNA expression (Fig. 7 ). ß-actin RNA was also assessed in all samples. Tyrosinase mRNA expression was normalized to the ß-actin mRNA. A positive cutoff point was considered as two standard deviations (luminosity units) above the mean of the healthy donor controls. In blood from 7 of 16 melanoma patients, luminosity was above the cutoff point. Healthy volunteer donors (Q,R,S,T, and U) were negative (Wilcoxon test; P = 0.01).

View larger version (31K): [in this window] [in a new window]   Figure 7. Assessment of tyrosinase mRNA expression in the blood of melanoma patients. Patient samples were analyzed on the Origen 1 Analyzer. To normalize specimens, each RNA sample was added to a RT-PCR reaction in a single tube and divided into duplicates. Primers specific for ß-actin were added to one tube, primers specific for tyrosinase were added to the second tube. Results are shown as luminosity (y-axis x 105) of tyrosinase mRNA relative to ß-actin, using the formula tyrosinase/actin x 105. Specimens A–P are RNAs isolated from blood obtained from melanoma patients. Specimens Q–U are RNAs isolated from healthy donor blood samples. Although not shown, the positive control of M12 melanoma cells was measured to have a luminosity of 4 x 105, using the same formula as specimens A–U. The dashed line represents the highest level of luminosity for healthy donor controls. Results 2SD above the mean of the healthy donor controls were considered positive.

	Discussion

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In summary, we have used ECL to detect the mRNA expression of the melanocyte/melanoma gene, tyrosinase, in an in vitro model system with M12 cells diluted in a nonmelanoma/melanocyte cells and in the blood of melanoma patients. In the in vitro model experiments, tyrosinase-specific RNA isolated from 1–1000 melanoma cells in a background of 10 B231 cells could be detected. In nested PCR reactions with 1 µg of RNA, murine leukemia virus reverse transcriptase, and AmpliTaq DNA polymerase, there were low luminescent signals for small numbers of melanoma cells. We improved the detection sensitivity substantially through the use of rTth for reverse transcription and amplification. One to 10 melanoma cells in 10 B231 cells could be detected in a single 40-cycle PCR reaction in three experiments. Detection also improved for the RT plus AmpliTaq polymerase reactions using 2 µg of RNA for cDNA synthesis and a single PCR amplification reaction. We were puzzled by these results because 40 rounds of amplification in the single reaction gave a higher signal than two 35-cycle amplification reactions in the nested PCR (total, 70 cycles). Several differences could account for this increase in sensitivity. One difference was that, to keep RNA concentrations equivalent in the AmpliTaq polymerase vs rTth polymerase reactions, 2 µg of RNA was used in 100-µL AmpliTaq polymerase reactions (in those using rTth polymerase, 1 µg of RNA was used in a 50-µL reaction volume). Another difference was that the single reactions were carried out using one outer and one nested primer from the two reaction protocols because only the nested primers were biotin-labeled. Yet another difference was that different amplification kits were used in these reactions, and AmpliTaq polymerase from different lots or manufacturers has been shown to lead to variability in amplification ability (14)(15). Increasing the amount of PCR product used in the nested PCR assay (e.g., 2, 5, or 10 µL) did not markedly improve detection (not shown).

Other studies involving PCR amplification of tyrosinase mRNA in melanoma cells, using these tyrosinase primers, have been described (5)(7)(8)(16). In three of these studies, nested primer sets were used in two rounds of amplification. The detection limit for melanoma cells varied in the individual studies. In the original study describing the HTYR 1–4 primers (5), the investigators could detect 1 melanoma cell in 5 x 10 leukocytes, 1 melanoma cell in 2 x 10 leukoyctes, or 1 melanoma cell in 2 mL of blood, depending on the melanoma cell line used. In the majority of the studies reported, the PCR cDNA products were detected after nested PCR by gel electrophoresis and ethidium bromide staining. The detection limit obtained with ECL was similar to those reported in these studies. However, the majority of the studies lack probe hybridization to verify the specificity of the cDNA product. The ECL system provided two advantages over the gel electrophoretic detection: It rapidly (<2 h for 49 samples) confirmed the identity of the product without time-consuming hybridization and detection or use of restriction enzyme mapping, and it quantified the PCR products (9)(17)(18)(19). Internal standards have been published for quantification (20)(21).

In several experiments, we detected luminescence in the B231 breast cancer RNA higher than values obtained for the negative control (no RNA added to the amplification reaction). Because oligo(dT) priming was not used for RT, illegitimate transcription (8)(22) of tyrosinase mRNA in the B231 breast cancer cell line may have contributed to the value of luminescence. Other investigators using these same primers and not oligo(dT) for RT also detected false-positive results when higher number of amplification cycles were implemented (5).

Melanoma patients in different clinical stages were assessed in a pilot study of 16 patients. Of seven positive patients, five had advanced stage disease. Previously, we have shown metastatic melanoma cells in the blood of early AJCC stage IIb patients (8). The frequency of detection of melanoma cells in blood by RT-PCR can vary between clinical stages, particularly in AJCC stages, II and III. Other factors such as sampling time, tumor burden and location, and treatment, play a role in the potential spread of tumor cells in blood.

The ECL system eliminates the necessity for the gel electrophoresis, transfer, hybridization, and detection protocols currently used for PCR analyses, thus saving time and labor costs. Because ECL uses hybridization-based procedures, specificity of the amplified product is confirmed, and the luminescence value readout system allows one to directly compare results from experiment to experiment through the use of internally amplified controls within the experiments. The ECL procedure is remarkably stable over a broad range of magnetic bead concentrations, probe concentrations, and probe hybridization conditions. This stability makes it extremely versatile in transferring protocols to different laboratories. The ease of use and time-saving features make it an attractive alternative for molecular detection of melanoma and other cancer cells that may play a role in diagnosis, detection of metastases, and the monitoring of response to therapy.

	Acknowledgments

 
We thank the following people for help in the preparation of the manuscript and for supplying reagents or advice for the experiments described in this report: Peter Korolkoff and Elena Katz (Perkin–Elmer) for expertise and assistance with the QPCR 5000 system, and J. Hereaux (IGEN) for sharing his considerable expertise on the Origen Analyzer. We thank the clinical staff of John Wayne Cancer Clinic for procuring blood from patients. This work was supported in part by grants CA 29605 and CA 1038 from the National Cancer Institute.

	Footnotes

 
¹ Nonstandard abbreviations: RT-PCR, reverse transcription-polymerase chain reaction; ECL, electrochemiluminescence; TBR, tris (2,2'-bipyridine) ruthenium (II); and AJCC, American Joint Committee on Cancer.

^{2 4} Certain commercial equipment, instruments, materials, or companies are identified in this paper to specify adequately the experimental procedure. Such identification does not imply recommendation or endorsement by the National Institute of Standards and Technology, nor does it imply that the materials or equipment identified are the best available for the purpose.

	References

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