Quantification of Melanoma mRNA Markers in Sentinel Nodes: Pre-Clinical Evaluation of a Single-Step Real-Time Reverse Transcriptase-Polymerase Chain Reaction Assay

Abstract

Although reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of melanocyte-associated mRNA can detect sentinel node melanoma metastases, most published assays are semi-quantitative methods of unknown sensitivity and precision, unsuitable for clinical use. We describe a single-step real-time quantitative RT-PCR assay for MART-1 and tyrosinase mRNAs, suitable for sentinel node analysis in a clinical setting. Using serial dilutions of melanoma cell line SK-MEL-28 RNA in water as a calibrator, we obtained linear calibration curves covering the range 0.5 to 10,000 arbitrary units (SK-MEL-28 melanoma cell equivalents). The sensitivity limit was 0.32 (MART-1) and 5 (tyrosinase) arbitrary units. Analytical imprecision was between 11% and 34%. MART-1 PCR efficiency was unaffected when samples were diluted with negative lymph node RNA rather than water, whereas tyrosinase PCR efficiency was halved. To evaluate the clinical suitability of our assay, we quantified melanocyte mRNAs in sentinel nodes with histologically verified micrometastases (n = 10) and benign nevus inclusions (n = 10), and in sentinel nodes without evidence of intranodal melanocytes (n = 10). We found significant differences in median melanocyte-derived mRNA levels comparing the three types of lymph nodes, suggesting that this quantitative molecular protocol may increase assay precision and be useful for the clinical evaluation of sentinel nodes.