Molecular staging of pathologically negative sentinel lymph nodes from melanoma patients using multimarker, quantitative real-time rt-PCR.

The aim of this study was to evaluate the prognostic potential of quantitative reverse-transcription, polymerase chain reaction (qRT-PCR) in melanoma patients with pathologically negative sentinel lymph nodes (SLN). Our study included 195 node-negative melanoma patients with a Breslow thickness greater than 0.76 mm (n = 158), or less than 0.76 mm but who had Clark level IV-V, microscopic ulceration, or pathological signs of regression (n = 32), and five patients with melanoma of unknown thickness. SLNs were examined by serial-section histopathology. A portion of each SLN was frozen for qRT-PCR analysis using markers Tyrosinase, MART1, SSX2, MAGEA3, PAX3, and GalNAc-T. In addition, two other markers (PLAB and L1CAM) were evaluated for melanoma specificity but not for SLN analysis. Median follow-up was 64 months, during which time there were 15 (7.7%) recurrences. A total of 370 lymph nodes were analyzed by qRT-PCR. No association was found between quantitative expression level of any marker and disease recurrence. Previously published primer designs were tested for PAX3 and GalNAc-T and revealed that alternative PAX3 transcripts are differentially expressed in melanoma and benign lymph nodes. No associations with recurrence were found regardless of the transcripts amplified by different primer sets. PLAB and L1CAM did not appear to differentiate between malignant melanoma and benign melanocytes or lymph nodes in our analysis. We conclude that, in this large cohort of patients, multimarker qRT-PCR analysis of SLNs did not correlate with disease recurrence. Our data support specific PAX3 splice variants but not GalNAc-T, PLAB or L1CAM as possible markers for melanoma metastasis to SLNs.