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Journal of Clinical Oncology, Vol 19, Issue 6 (March), 2001: 1723-1727
© 2001 American Society for Clinical Oncology

Interlaboratory Evaluation of a New Reverse Transcriptase Polymerase Chain Reaction–Based Enzyme-Linked Immunosorbent Assay for the Detection of Circulating Melanoma Cells: A Multicenter Study of the Dermatologic Cooperative Oncology Group

By U. Reinhold, C. Berkin, A.-K. Bosserhoff, A. Deutschmann, C. Garbe, R. Gläser, R. Hein, G. Krähn, R. U. Peter, G. Rappl, B. Schittek, S. Seiter, S. Ugurel, M. Volkenandt, W. Tilgen

From the Department of Dermatology, The Saarland University Hospital, Homburg/Saar; Department of Pathology, University of Aachen, Aachen; Department of Dermatology, University of Kiel, Kiel; Department of Dermatology, Ludwig-Maximilians-University of Munich; Department of Dermatology, Technical University Munich, Munich; Department of Dermatology, University of Tübingen, Tübingen; and Department of Dermatology, University of Ulm, Ulm, Germany.

Address reprint requests to Uwe Reinhold, MD, Department of Dermatology, The Saarland University Hospital, 66421 Homburg/Saar, Germany; email: uwe.reinhold{at}med-rz.uni-sb.de


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIAL AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
PURPOSE: Reverse transcription-polymerase chain reaction (RT-PCR)–based detection of tyrosinase mRNA is the most frequently used laboratory method for the detection of circulating tumor cells in melanoma patients. However, previously published results showed considerable variability in the PCR positivity rates.

MATERIALS AND METHODS: We designed a collaborative study to assess the sensitivity, specificity, and clinical relevance of a new standardized RT-PCR–based enzyme-linked immunosorbent assay (ELISA) for the detection of circulating melanoma cells. Blood samples of healthy donors mixed with cells of a melanoma cell line were prepared in a blinded fashion, and aliquots were sent to seven participating laboratories experienced in RT-PCR.

RESULTS: The results demonstrate a high sensitivity (1 melanoma cell/mL blood) and specificity (no false-negatives and 7.4% [2 of 28] false-positives) of the assay and a satisfactory rate of interlaboratory reproducibility. The analysis of aliquots of blinded samples derived from 60 melanoma patients identified tyrosinase mRNA in 17 of 60 (28.3%): three (20%) of 15 stage I patients, two (13.3%) of 15 stage II patients, five (35.7%) of 14 stage III patients, and seven (43.8%) of 16 stage IV patients. The interlaboratory reproducibility of positive samples, however, was extremely low and indicates the presence of low amounts of target mRNA.

CONCLUSION: Reverse transcriptase-PCR ELISA has a high sensitivity and specificity for the detection of tyrosinase mRNA in peripheral blood cells. The low interlaboratory reproducibility for the detection of tumor cells in blood samples of melanoma patients, however, raises the question of relevance of this assay for clinical use.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIAL AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
THE NESTED reverse transcription-polymerase chain reaction (RT-PCR) is a highly sensitive procedure used to detect tyrosinase mRNA, which is expressed in melanoma cells but not in normal peripheral blood cells. The presence of tyrosinase mRNA is therefore taken as an indicator for circulating tumor cells in the blood of melanoma patients.1 A large number of studies using the detection of tyrosinase mRNA by RT-PCR in the peripheral blood of melanoma patients have been published in the last few years ( Table 1).2-21 The clinical relevance of this procedure, however, still remains unclear inasmuch as the results of different groups have varied considerably. Even in melanoma patients with distant metastases, the data on the percentage of individuals with evidence of tyrosinase mRNA in their blood varied from 0% to 100%.2,12-14 These differences may be caused by variations in sample processing, RNA extraction, or PCR amplification, resulting in lower sensitivity, or even by problems of contamination. To assess this question, a quality-assurance initiative of the European Organization for Research and Treatment of Cancer Melanoma Cooperative Group for tyrosinase RT-PCR analysis was initiated. Nine laboratories that participated in this trial confirmed that unacceptable specificity or sensitivity clearly plays a role in the discrepant results and thus fosters difficulties in interpretation of tyrosinase RT-PCR data.15 These data emphasized the need of a standardized procedure for the detection of tyrosinase mRNA. Such a procedure would allow data comparison and possible future clinical application of this method in melanoma patients. Therefore we tested a new approach for the detection of tyrosinase mRNA in peripheral blood of melanoma patients on the basis of a standardized procedure for blood sample stabilization and mRNA isolation,16 followed by a standardized technique for RT-PCR amplification of the tyrosinase transcript and enzyme-linked immunosorbent assay (ELISA) detection of the PCR product. The goal of our study was to evaluate the reliability and clinical relevance of this standardized and commercially available RT-PCR ELISA for the detection of tumor cells in peripheral blood of melanoma patients.


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  		Table 1. Comparison of Studies Using RT-PCR for the Detection of Circulating Melanoma Cells
 

   MATERIAL AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIAL AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Patients and Sample Preparation
After obtaining informed consent, 35 mL of heparinized blood was obtained from four normal healthy control subjects and 60 patients with malignant melanoma (28 men and 32 women; age, 29 to 84 years). Clinical stage was defined according to the American Joint Committee on Cancer/International Union Against Cancer criteria: stage I and stage II included patients with primary melanoma; stage III included patients with regional lymph node or in-transit metastases; and stage IV included patients with distant metastases. Follow-up was performed at least in 3-month intervals including physical examination, x-ray of the chest, ultrasound of the abdomen and lymph nodes, and blood chemistry. Patients in advanced stages of disease (stages III and IV) additionally underwent computer tomography of the brain and scintigraphy of the skeleton. To evaluate the sensitivity, specificity, and reproducibility of this approach, 35 mL of heparinized whole blood samples from healthy control subjects were spiked with defined amounts of MelJu melanoma cells (0, 35, 35 x 101, 35 x 102, 35 x 103, and 35 x 104). To evaluate the intralaboratory reproducibility, two different series of identically spiked blood samples were prepared. An additional negative control panel consisted of blood samples from four different healthy donors. Each of the 35-mL blood samples was divided into seven identical aliquots of 5 mL that were immediately mixed with 45 mL of RNA/DNA Stabilization Reagent for Blood/Bone Marrow (Roche Diagnostics GmbH, Penzberg, Germany), resulting in simultaneous lysis of cells and immediate stabilization of the nucleic acids contained therein. Stabilized blood samples were stored at -70°C. To assure constant quality, all of the samples were prepared in the laboratory at the Department of Dermatology, University of Homburg/Saar, Germany, and distributed as blinded samples by overnight shipment on dry ice to the participating laboratories.

RNA Isolation and RT-PCR ELISA
The same procedure for RNA extraction, RT-PCR amplification, and detection of the tyrosinase mRNA transcript was performed in each participating laboratory according to a standardized instruction manual. Poly(A)-mRNA was purified using the mRNA Isolation Kit for Blood/Bone Marrow as described by the manufacturer (Roche Diagnostic Company). The entire mRNA purified from each sample was directly used for cDNA synthesis and subsequent PCR reaction. The Tyrosinase RT-PCR ELISA (Roche Diagnostic Company) was used for RT-PCR amplification and detection of the tyrosinase transcript according to the manufacturer’s instructions. The test kit included a positive control containing mRNA isolated from MelJu melanoma cells and a negative control containing double-distilled water. Furthermore, the integrity of the mRNA of each sample was verified by RT-PCR with primers for ß2-microglobulin. In the first step, parallel one-step reactions were performed combining the reverse transcription of tyrosinase or ß2-microglobulin mRNA into cDNA and the amplification of the cDNA by PCR. The reverse transcriptase avian myeloblastosis virus was specifically primed using a biotin-labeled oligonucleotide. The PCR reaction was catalyzed by Taq DNA polymerase contained in the enzyme mixture and resulted in a 284-bp PCR product for tyrosinase and a 119-bp amplicon for ß2-microglobulin. In a second step, the products for tyrosinase generated during the first RT-PCR were further amplified using a pair of nested primers and Taq DNA polymerase. One of the nested primers was labeled with biotin. The tyrosinase-derived nested PCR product was 207 bp in size. In a third step, PCR products were denatured and hybridized to a digoxigenin (DIG)-labeled detection probe specific for part of the target sequence. The resulting hybrid was immobilized via biotin label to a streptavidin-coated microtiter plate. The immobilized amplicon was subsequently detected photometrically using an antibody to DIG coupled to horseradish peroxidase (anti-DIG-HRP) and the sensitive peroxidase substrate tetramethylbenzidine. The absorbance of the negative controls for tyrosinase and ß2-microglobulin had to be less than 0.2 A450 nm to A690 nm. The absorbance of the positive controls for tyrosinase and ß2-microglobulin had to be greater than 1.0 A450nm to A690nm. Samples were regarded as tyrosinase- or ß2-microglobulin-positive if the difference in absorbance is greater than 0.2 A450nm to A690nm units.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIAL AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Sensitivity, Specificity, and Reproducibility
The primary goal of the study was to evaluate the sensitivity, specificity, and reproducibility of a standardized procedure for the detection of tyrosinase mRNA. Therefore, we prepared blood samples from healthy donors spiked with different amounts of MelJu melanoma cells, which were split into seven identical 5-mL aliquots and immediately mixed with 45 mL of a standardized stabilization reagent. Blood samples from four different healthy control subjects were used as negative controls. Aliquots of these blinded samples were sent to seven different laboratories for the evaluation of tyrosinase mRNA transcripts. Our analysis demonstrated that all laboratories had reported correct results for blood samples mixed with melanoma cells (Table 2). The intralaboratory reproducibility was confirmed by identical results generated from two identically spiked test panels. The detection limit was 1 melanoma cell in 1 mL of blood in all seven participating laboratories, thus providing a sensitivity of approximately 1 tumor cell per 106 peripheral blood mononuclear cells. No false-negative results were reported. For all 28 control samples prepared from four different healthy control subjects, only two false-positive samples were reported in two different laboratories. Blood mRNA integrity was confirmed in all samples by RT-PCR with primers for ß2-microglobulin. In addition, all laboratories reported correct results for internal positive (containing mRNA isolated from a melanoma cell line) and negative controls (containing double-distilled water) of the RT-PCR-ELISA.


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  		Table 2. Interlaboratory Sensitivity and Specificity of MelJu Cell Detection in Spiked Whole Blood Samples by Tyrosinase RT-PCR ELISA
 
Analysis of Blood Samples From Melanoma Patients
After having verified sensitivity and specificity, the second phase of our multicenter evaluation trial focused on possible clinical significance of this procedure using the blood samples obtained from melanoma patients. The blinded test panel consisted of 420 aliquots prepared from blood samples of 60 patients with melanoma in different clinical stages. The samples were sent to the seven participating laboratories and subsequently tested for the presence of tyrosinase mRNA as described previously.

The results of all testing performed in the seven participating laboratories are summarized in Table 3. Seventeen (28.3%) of all 60 patients were tested positive for tyrosinase mRNA in at least one of the participating laboratories. These 17 patients consisted of three (20.0%) of 15 patients with stage I disease, two (13.3%) of 15 patients with stage II disease, five (35.7%) of 14 patients with stage III disease, and seven (43.8%) of patients with 16 stage IV disease.


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  		Table 3. Overall Results and Distribution of Patients Tumor Stage for RT-PCR ELISA
 
The interlaboratory reproducibility of positive tyrosinase results in blood samples of these melanoma patients, however, was extremely low ( Table 4). Only 18 (4.3%) of all 420 blood samples from 60 different melanoma patients were found to be positive for tyrosinase mRNA. Sixteen patients tested positive in only one of seven participating laboratories. One patient was found to be positive in two different laboratories. The remaining laboratories reported negative results for all test samples even though integrity of mRNA was confirmed by the presence of ß2-microglobulin mRNA.


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  		Table 4. Interlaboratory Reproducibility of Tyrosinase mRNA Detection by RT-PCR ELISA in 420 Blinded Aliquots of 60 Melanoma Patient Blood Samples
 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIAL AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Reverse transcription of tumor-specific mRNA and cDNA amplification using PCR techniques have been reported to facilitate the early detection of circulating tumor cells in melanoma patients.1,2,5,6,8,17,18 Currently, tyrosinase is the most commonly used mRNA marker tested for in melanoma. Despite the large number of studies performed, the significance and clinical value of this method for the detection of tumor dissemination in melanoma patients are still unclear.12-14 To date more than 30 studies have been published on this topic, with the numbers of patients positive for tyrosinase mRNA in the peripheral blood varying from 0% to 100% (Table 1). Numerous factors may have contributed to these reported inconsistencies. They may include differences in RNA extraction methods and variations in thermal cycling techniques, as well as the possibility of cross contamination.19 Furthermore, biologic factors, including the possibility of intermittent shedding of tumor cells into the bloodstream or a low level or absence of tyrosinase mRNA expression in subclones of melanoma cells, have to be taken into account.14,21 The quality-assurance initiative devised by the European Organization for Research and Treatment of Cancer Melanoma Cooperative Group to assess these questions reported that four of nine laboratories performing tyrosinase RT-PCR had demonstrated an unacceptable specificity or sensitivity when evaluated by a blinded interlaboratory study.15 The authors suggest that sample preparation, RNA extraction, and cDNA synthesis, rather than the PCR protocols, may account for most of the heterogeneity in results when using tyrosinase RT-PCR assays. A number of standardization issues, therefore, appear to be most important to enhance the comparability of results of the tyrosinase RT-PCR assay.

With the need for a reliable marker for malignant melanoma and furthermore the fact that certain laboratories had already started using this technique for patient purposes, a standardized reliable procedure was needed. The present interlaboratory study was performed to evaluate the applicability and reliability of a new standardized procedure for the detection of circulating tumor cells from peripheral blood of melanoma patients. In contrast to previously performed techniques, the procedure used in this study was mostly based on standardized procedures. The following features are characteristic of the procedure and had not been used in the detection of tyrosinase mRNA before: (1) the use of an RNA stabilization reagent simplifies the handling of blood samples and allows freezing and storing the blood lysate right after addition of the stabilization reagent, thus immediately preventing nucleic acids from degradation; (2) density gradient centrifugations are not necessary, therefore avoiding the possible loss of tumor cells during this step; (3) a standardized procedure for the purification of poly(A)-mRNA is used to enhance sensitivity by enriching target mRNA and, by eliminating inhibitory factors, results in high concentrations of total RNAs and chromosomal DNA in the mRNA preparation; and (4) RT-PCR amplification and detection of the tyrosinase transcript is performed by a standardized RT-PCR ELISA procedure. The primary aim of the study was to evaluate the sensitivity, specificity, and reproducibility of the RT-PCR-ELISA for tyrosinase mRNA detection. All participating laboratories detected tyrosinase transcripts in blood samples mixed with MelJu melanoma cells with the same sensitivity at 1 cell per 1 mL blood. No false-negative results were reported, and the rate of false positives was 7.1%, giving a specificity rate of 92.9%. The results indicate that the RT-PCR ELISA in combination with a standardized mRNA isolation method can achieve a high and reproducible level of sensitivity and specificity of detection of tyrosinase mRNA in blood samples that is comparable to other RT-PCR–based assays.

By using exactly the same procedure for mRNA isolation and detection of tyrosinase transcripts by RT-PCR ELISA, we then tested identical aliquots of blinded blood samples from 60 melanoma patients in seven different laboratories. Tyrosinase mRNA was detected in the peripheral blood of 17 of 60 patients, and the number of tyrosinase mRNA–positive patients showed a positive correlation with the stage of the disease. This PCR positive rate is similar to the one reported by Schittek et al18 and Mellado et al6 but higher than that of other studies reporting a lower rate of PCR-positive results in stage I and II patients.13,14,21 The most interesting finding of the present study, however, is the fact that the reproducibility of PCR-positive results of the same blood sample tested in different laboratories is extremely low. These differences in reproducibility could not be explained by differences in sample processing, mRNA quality, and PCR methods. The results suggest that the poor reproducibility testing the blood of patients is most likely caused by low amounts of melanoma-specific transcripts present in the blood that seem to be lower than 1 MelJu equivalent per milliliter of blood in tested melanoma patients. In the present study, blood samples from melanoma patients were divided before mRNA extraction. It seems possible that blood samples containing few tumor cells were divided into a certain percentage of aliquots that was negative for the sample by the laws of probability distribution. The results of our study indicate that low amounts of tyrosinase mRNA in the peripheral blood is the most likely reason for the large discrepancies described in the frequency of circulating melanoma cells. This assumption is further supported by reports indicating a low reproducibility of PCR-positive blood samples after repeated analyses of the sample.4,18,22 Furthermore, real-time quantitative RT-PCR showed that the low reproducibility was mainly caused by a low number of target molecules.22 Although we could demonstrate that the detection of tyrosinase transcripts by the RT-PCR ELISA procedure used in this interlaboratory study is a sensitive and reproducible method, the results obtained from samples obtained from melanoma patients indicate that the clinical relevance of this assay for monitoring micrometastatic disease is questionable.


   ACKNOWLEDGMENTS
 
Supported by Roche Diagnostics, Penzberg, Germany.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIAL AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
1. Smith B, Selby P, Southgate J, et al: Detection of melanoma cells in peripheral blood by means of reverse transcriptase and polymerase chain reaction. Lancet 338: 1227-1229, 1991[Medline]

2. Brossart P, Keilholz U, Willhauck M, et al: Hematogenous spread of malignant melanoma cells in different stages of disease. J Invest Dermatol 101: 887-889, 1993[Medline]

3. Brossart P, Keilholz U, Scheibenbogen C, et al: Detection of residual tumor cells in patients with malignant melanoma responding to immunotherapy. J Immunother 15: 38-41, 1994

4. Farthmann B, Eberle J, Krasagakis K, et al: RT-PCR for tyrosinase-mRNA-positive cells in peripheral blood: Evaluation strategy and correlation with known prognostic markers in 123 melanoma patients. J Invest Dermatol 110: 263-265, 1998[Medline]

5. Mellado B, Colomer D, Castel T, et al: Detection of circulating neoplastic cells by reverse-transcriptase polymerase chain reaction in malignant melanoma: Association with clinical stage and prognosis. J Clin Oncol 14: 2091-2097, 1996[Abstract/Free Full Text]

6. Mellado B, Gutierrez L, Castel T, et al: Prognostic significance of the detection of circulating malignant cells by reverse transcriptase-polymerase chain reaction in long-term clinically disease-free melanoma patients. Clin Cancer Res 5: 1843-1848, 1999[Abstract/Free Full Text]

7. Ghossein RA, Coit D, Brennan M, et al: Prognostic significance of peripheral blood and bone marrow tyrosinase messenger RNA in malignant melanoma. Clin Cancer Res 4: 419-428, 1998[Abstract/Free Full Text]

8. Curry BJ, Myers K, Hersey P: Polymerase chain reaction detection of melanoma cells in the circulation: Relation to clinical stage, surgical treatment, and recurrence from melanoma. J Clin Oncol 16: 1760-1769, 1998[Abstract]

9. Battayani Z, Grob JJ, Xerri L, et al: Polymerase chain reaction detection of circulating melanocytes as a prognostic marker in patients with melanoma. Arch Dermatol 131: 443-447, 1995[Abstract/Free Full Text]

10. Pittman K, Burchill S, Smith B, et al: Reverse transcriptase-polymerase chain reaction for expression of tyrosinase to identify malignant melanoma cells in peripheral blood. Ann Oncol 7: 297-301, 1996[Abstract/Free Full Text]

11. Jung FA, Buzaid AC, Ross MI, et al: Evaluation of tyrosinase mRNA as a tumor marker in the blood of melanoma patients. J Clin Oncol 15: 2826-2831, 1997[Abstract]

12. Foss AJ, Guille MJ, Occleston NL, et al: The detection of melanoma cells in peripheral blood by reverse transcription-polymerase chain reaction. Br J Cancer 72: 155-159, 1995[Medline]

13. Gläser R, Rass K, Seiter S, et al: Detection of circulating melanoma cells by specific amplification of tyrosinase complementary DNA is not a reliable tumor marker in melanoma patients: A clinical two-center study. J Clin Oncol 15: 2818-2825, 1997[Abstract]

14. Reinhold U, Ludtke-Handjery HC, Schnautz S, et al: The analysis of tyrosinase-specific mRNA in blood samples of melanoma patients by RT-PCR is not a useful test for metastatic tumor progression. J Invest Dermatol 108: 166-169, 1997[Medline]

15. Keilholz U, Willhauck M, Rimoldi D, et al: Reliability of reverse transcription-polymerase chain reaction (RT-PCR)-based assays for the detection of circulating tumour cells: A quality-assurance initiative of the EORTC Melanoma Cooperative Group. Eur J Cancer 34: 750-753, 1998

16. Noppen C, Luescher U, Zuber M, et al: Detection of tumor-associated antigen gene expression in peripheral blood by RT-PCR in combination with the mRNA Isolation Kit for Blood/Bone Marrow. Biochemica 4: 11-13, 1997

17. Kunter U, Buer J, Probst M, et al: Peripheral blood tyrosinase messenger RNA detection and survival in malignant melanoma. J Natl Cancer Inst 88: 590-594, 1996[Abstract/Free Full Text]

18. Schittek B, Bodingbauer Y, Ellwanger U, et al: Amplification of MelanA messenger RNA in addition to tyrosinase increases sensitivity of melanoma cell detection in peripheral blood and is associated with the clinical stage and prognosis of malignant melanoma. Br J Dermatol 141: 30-36, 1999[Medline]

19. Schittek B, Blaheta H-J, Flörchinger G, et al: Increased sensitivity for the detection of malignant melanoma cells in peripheral blood using an improved protocol for reverse transcription-polymerase chain reaction. Br J Dermatol 141: 37-43, 1999[Medline]

20. Hoon DS, Wang Y, Dale PS, et al: Detection of occult melanoma cells in blood with a multiple-marker polymerase chain reaction assay. J Clin Oncol 13: 2109-2116, 1995[Abstract/Free Full Text]

21. Stevens GL, Scheer WD, Levine EA: Detection of tyrosinase mRNA from the blood of melanoma patients. Cancer Epidemiol Biomarkers Prev 5: 293-296, 1996[Abstract]

22. de Vries TJ, Fourkour A, Punt CJ, et al: Reproducibility of detection of tyrosinase and MART-1 transcripts in the peripheral blood of melanoma patients: A quality control study using real-time quantitative RT-PCR. Br J Cancer 80: 883-891, 1999[Medline]

Submitted March 30, 2000; accepted December 7, 2000.


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