Detection of occult melanoma cells in paraffin-embedded histologically negative sentinel lymph nodes using a reverse transcriptase polymerase chain reaction assay

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 sent inel lymph node (SLN) has been proposed as a reliable predictor of metastat ic disease in the lymphatic basin draining the primary melanoma. In this st udy, we screened both paraffin-embedded SLNs and peripheral-blood (PB) samp les from MM patients at various stage of disease using a multimarker revers e transcriptase polymerase chain reaction (RT-PCR) assay. The prognostic si gnificance of the presence of PCR-positive markers was also evaluated. Patients and Methods: Total RNA was obtained from paraffin-embedded SLN sec tions and PB samples of 75 MM patients. RT-PCR was performed using tyrosina se and MelanA/MART1 as melanoma-associated markers. Radiolabeled PCR produc ts were analyzed on denaturing polyacrylamide gels. Results: Good sensitivity of the RT-PCR assay on archival tissues was demon strated 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 micrometastas es, thus improving staging of patients with melanoma. J Clin Oncol 19:1437- 1443. (C) 2001 by American Society of Clinical Oncology.