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

• Published in: Journal of clinical oncology Vol. 19; no. 6; pp. 1723 - 1727
• Main Authors: REINHOLD, U, BERKIN, C, SCHITTEK, B, SEITER, S, UGUREL, S, VOLKENANDT, M, TILGEN, W, BOSSERHOFF, A.-K, DEUTSCHMANN, A, GARBE, C, GLÄSER, R, HEIN, R, KRÄHN, G, PETER, R. U, RAPPL, G
• Format: Journal Article
• Language: English
• Published: Baltimore, MD American Society of Clinical Oncology 15.03.2001; Lippincott Williams & Wilkins
• Subjects: Adult; Aged; Aged, 80 and over; Biological and medical sciences; Dermatology; DNA, Neoplasm - analysis; Enzyme-Linked Immunosorbent Assay - methods; Enzyme-Linked Immunosorbent Assay - standards; Female; Humans; Investigative techniques, diagnostic techniques (general aspects); Medical sciences; Melanoma - diagnosis; Melanoma - pathology; Middle Aged; Neoplastic Cells, Circulating; Observer Variation; Pathology. Cytology. Biochemistry. Spectrometry. Miscellaneous investigative techniques; Reproducibility of Results; Reverse Transcriptase Polymerase Chain Reaction - standards; Sensitivity and Specificity; Skin Neoplasms - diagnosis; Skin Neoplasms - pathology; Human; Skin disease; RNA-directed DNA polymerase; Enzyme; Transferases; Melanoma; Malignant tumor; Laboratory; Enzyme immunoassay; Blood; Polymerase chain reaction; Nucleotidyltransferases; Sensitivity; Specificity; Skin; Diagnosis; Molecular biology; Reliability; ELISA assay
• ISSN: 0732-183X; 1527-7755
• DOI: 10.1200/JCO.2001.19.6.1723

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. 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. 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. 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.