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American Journal of Pathology, Vol 149, 759-764, Copyright © 1996 by American Society for Investigative Pathology

REGULAR ARTICLES

Molecular test for the detection of tumor cells in blood and sentinel nodes of melanoma patients

D Van der Velde-Zimmermann, JF Roijers, A Bouwens-Rombouts, RA De Weger, PW De Graaf, MG Tilanus and JG Van den Tweel
Department of Pathology, University Hospital, Utrecht, The Netherlands.

Lymph node metastasis dramatically decreases the 5-year survival of melanoma patients. The so-called sentinel node surgery offers a therapeutic approach to resect the first draining lymph node. This technique enables accurate staging of melanoma patients in an early stage of the disease. Detection of a sentinel node metastasis is a strong argument for local lymphadenectomy. To improve the detection of micrometastasis in sentinel nodes of melanoma patients, molecular biological techniques were used. The exclusively melanocyte-specific tyrosinase transcript was amplified by reverse transcription followed by polymerase chain reaction (RT-PCR). In sentinel node examination, the detection of tyrosinase-positive cells by RT-PCR was compared with routine immunohistochemistry. From 16 patients, a total of 28 lymph nodes were tested. The lymph nodes were derived after lymphadenectomy (4 patients) and after sentinel node resection (12 patients with melanoma stage I). By using RT-PCR we could detect 100 tumor cells in a background of 10(8) peripheral blood mononuclear cells. The negative controls were all negative for tyrosinase. Cryostat sections of lymph nodes for mRNA isolation were alternated with sections for immunohistochemistry. By using tyrosinase RT-PCR, we detected 6 additional positive sentinel nodes in patients with melanoma stage I. Furthermore, the tyrosinase RT-PCR enabled us to design a blood test for circulating melanoma cells. Therefore, mRNA was directly isolated from whole blood of 23 blood samples, of which 3 samples were positive for tyrosinase. The present study demonstrates the possibility of a simple and rapid blood test for melanoma patients that has not been available until now. Furthermore, the detection of micrometastasis in sentinel nodes by tyrosinase RT-PCR dramatically increases the accuracy of melanoma staging.

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