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Detection of Occult Melanoma Cells in Paraffin-Embedded Histologically Negative Sentinel Lymph Nodes Using a Reverse Transcriptase Polymerase Chain Reaction Assay

From the Institute of Molecular Genetics, National Research Council of Italy, Alghero (SS); the Institute of Pathology, University of Sassari, Sassari; the National Tumor Institute "Fondazione G. Pascale"; and the Department of Dermatology, Second University of Naples, Naples, Italy

Address reprint requests to Giuseppe Palmieri, MD, Institute of Molecular Genetics, National Research Council of Italy, Alghero (SS), Casella Postale, 07040 Santa Maria La Palma (Sassari), Italy; email: gpalmieri{at}yahoo.com

PURPOSE: Detection of occult metastasis before the development of clinical disease could allow more accurate staging, appropriate follow-up procedures, and adjuvant therapies in patients with malignant melanoma (MM). The sentinel lymph node (SLN) has been proposed as a reliable predictor of metastatic disease in the lymphatic basin draining the primary melanoma. In this study, we screened both paraffin-embedded SLNs and peripheral-blood (PB) samples from MM patients at various stage of disease using a multimarker reverse transcriptase polymerase chain reaction (RT-PCR) assay. The prognostic significance of the presence of PCR-positive markers was also evaluated.

PATIENTS AND METHODS: Total RNA was obtained from paraffin-embedded SLN sections and PB samples of 75 MM patients. RT-PCR was performed using tyrosinase and MelanA/MART1 as melanoma-associated markers. Radiolabeled PCR products were analyzed on denaturing polyacrylamide gels.

RESULTS: Good sensitivity of the RT-PCR assay on archival tissues was demonstrated after comparison of RT-PCR results on frozen and paraffin-embedded SLNs from 16 MM patients. Significant correlation between the disease stage and marker expression in both PB and SLN samples was observed; the highest value was for patients who were positive for both markers in SLN (P = .006). Progression of disease was significantly associated with the total number of PCR-positive markers in both PB (P = .034) and SLN (P = .001) samples.

CONCLUSION: Although sensitivity is lowered by the use of paraffin-embedded specimens, our data indicate that RT-PCR analysis of serial sections from archival SLNs may be helpful in improving detection of occult micrometastases, thus improving staging of patients with melanoma.

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