Accurate molecular detection of melanoma nodal metastases: an assessment of multimarker assay specificity, sensitivity, and detection rate

Abstract

Background: The application of lymphoscintigraphy followed by sentinel lymph node (SN) biopsy to patients with primary melanoma has revolutionised the ability to identify accurately, yet conservatively, those patients who harbour occult nodal metastases. The molecular detection of SN micrometastases facilitates the cost effective analysis of the entire SN using multiple markers. Currently, a lack of marker specificity is the main barrier preventing the molecular evaluation of SN tissue from becoming clinically applicable.

Aims: To develop a reproducible multimarker reverse transcription-polymerase chain reaction (RT-PCR) assay, with the emphasis on achieving high specificity for the accurate detection of melanoma metastases in nodal tissue.

Methods: Three pigment cell specific (PCS) markers-tyrosinase, Pmel-17, and MART-1-and one cancer testis antigen (CTA)-MAGE-3-were selected for use in a multimarker RT-PCR assay. The conditions for this assay were optimised.

Results: High specificity was achievable for each marker by optimising the PCR cycle number such that unwanted transcripts (that is, illegitimate transcripts and/or specific transcripts from other low abundance nodal cell types) remained undetectable in appropriate controls (normal visceral nodes). Tyrosinase was 100% specific at 40 PCR cycles, MAGE-3 and MART-1 at 35 PCR cycles, and Pmel-17 at 30 PCR cycles. Tyrosinase proved to be the most sensitive marker, detecting 10 melanoma cells in 0.1 g of nodal tissue.

Conclusions: Excellent reproducibility of the entire nodal processing and RT-PCR protocol for the detection of very low numbers of melanoma cells in nodal tissue was shown, although there is a risk of false positives using the PCS markers alone, because of an approximate 4-8.5% incidence rate of nodal nevi in melanoma draining SNs (these nevi being absent in all other normal nodes). MAGE-3 was shown to be the only marker that is not expressed by melanocytes. However, because not all melanomas express MAGE-3, it is recommended that more emphasis should be placed on the development of a panel of CTA markers to ensure a zero false positive rate and to provide optimum detection.

Figure 1

Optimisation of the PCR cycle number for each marker. Lanes 1–10, 10 normal (donor) lymph nodes. Lanes +C, positive controls (UCT-Mel-1) for each marker. The optimal PCR cycle number for tyrosinase, MAGE-3, MART-1, Pmel-17, and porphobilinogen deaminase (PBGD) was 40, 35, 35, 30, and 30, respectively. Using higher PCR cycle numbers than these for each marker resulted in the detection of unwanted transcripts. The PCR product sizes for tyrosinase, MAGE-3, MART-1, Pmel-17, and PBGD are 250, 413, 299, 669, and 339 bp, respectively. The results are representative of two independent experiments.

Figure 2

Assessing the sensitivity of each marker by using normal nodal tissue spiked with decreasing numbers of UCT-Mel-1 cells. Lanes 1 to 4: 10¹, 10², 10³, and 10⁴ melanoma cells spiked into 0.1 g of normal nodal tissue, respectively. The results are representative of two independent experiments. PBGD, porphobilinogen deaminase.

Figure 3

Reproducibility of the entire protocol used in our study. Lanes 1–4, tyrosinase and porphobilinogen deaminase (PBGD) yields from four independent nodal tissue samples each spiked with 10 UCT-Mel-1 cells and subjected to the entire protocol (nodal processing and reverse transcription-polymerase chain reaction (RT-PCR)). Lane 5, negative controls (H₂O) for tyrosinase and PBGD. Lane 6, positive controls for tyrosinase and PBGD (UCT-Mel-1 RNA as RT template). The results are representative of two independent RT-PCR assays for each spiked node.

Figure 4

Assessing the in vitro and in vivo detection rates for each marker. (A) Lanes 1–8, UCT-Mel-1 to UCT-Mel-8 cell lines, respectively. (B) Lanes 1–4, melanoma involved lymph nodes numbers 1 to 4, respectively. MART-1 and MAGE-3, in addition to Pmel-17 and porphobilinogen deaminase (PBGD) were multiplexed. The results are representative of two independent experiments.

Figure 5

Differentiating between melanoma cells and melanocytic nevi. Lanes A and B, melanoma involved node and pigmented skin, respectively. The results are representative of two independent experiments. PBGD, porphobilinogen deaminase.