DETECTION OF SUBMICROSCOPIC LYMPH-NODE METASTASES WITH POLYMERASE CHAIN-REACTION IN PATIENTS WITH MALIGNANT-MELANOMA

Background The presence or absence of lymph node metastases in patient s with malignant melanoma is the most powerful prognostic factor for p redicting survival. ii regional nodal metastases are found, the 5-year survival for the patient decreases approximately 50%. If the presence or absence of regional nodal metastases will determine which patients receive formal dissections or which patients enter adjuvant trials, t hen a technique is needed to accurately screen lymph node samples for occult disease. Routine histopathologic examination routinely underest imates the number of patients with metastases. This study was initiate d to develop a highly sensitive clinically applicable method to detect micrometastases by examining lymph nodes for the presence of tyrosina se messenger RNA (mRNA). The hypothesis was that if mRNA for tyrosinas e is found in the lymph node preparation, that finding is good evidenc e that metastatic melanoma cells are present. Methods The assay is acc omplished using the combination of reverse transcription and double-ro und polymerase chain reaction (RT-PCR). The amplified samples are exam ined on a 2% agarose gel and tyrosinase cDNA is seen as a 207 base pai r fragment. Lymph node preparations from 29 patients who were clinical ly stage I and II and undergoing elective node dissections were analyz ed both by standard pathologic staining and RT-PCR. Results Eleven of 29 lymph node (38%) samples from 29 patients with intermediate thickne ss melanoma were pathologically positive. Nineteen of the 29 lymph nod e preparations (66%) were RT-PCR-positive, and these included all of t he pathologically positive samples, so that the false-negative rate wa s 0. In a spiking experiment, one SK-Mel-28 melanoma cell in a backgro und of one million normal lymphocytes could be detected, thus indicati ng the sensitivity of this method. In addition, analysis by restrictio n enzyme mapping showed that the amplified 207-bp PCR product produced is part of the tyrosinase gene sequence. Conclusion The RT-PCR method is an extremely sensitive, reproducible, and efficient technique for the identification of micrometastases in patients with melanoma and co uld be widely applicable. If clinical correlation is obtained, staging of the melanoma patient becomes more accurate, and treatment becomes more standardized and rational, because all those patients who have ev idence of nodal disease can be identified so that they may benefit fro m more extensive surgery (formal node dissections) or adjuvant therapi es, Based on these results, RT-PCR could be a powerful tool to detect micrometastatic melanoma.