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Chromosomal instability is a well-described feature of malignant tumors. Melanomas have typical patterns of chromosomal instability compared with benign nevi, which have minimal DNA copy number change. A few malignant melanomas and their benign counterparts, nevi, prove difficult to diagnose on histopathologic analysis alone, which is currently the gold standard. Quantitative PCR–based assays called duplex ratio tests (DRTs) have been developed by our laboratory for application using DNA from FFPE samples of melanomas and nevi. The reproducibility and accuracy of the DRTs were demonstrated and appropriate correction factors for DNA quality calculated for each assay, based on the results of 108 diploid samples. As a panel, seven DRTs were able to differentiate unambiguous cases of melanoma and nevi with a sensitivity of 87% (95% CI, 83%–91%) and a specificity of 88% (95% CI, 84%–92%) in a series of 145 melanomas and 123 nevi. The DRT scores for 20 nonmetastasizing primary melanomas and 20 metastasizing primary melanomas revealed that DRTs had a marginal benefit as prognostic markers. DRTs have early potential to act as molecular biomarkers of melanoma on FFPE specimens pending validation, and DRTs may have applicability as prognostic markers in melanoma or other tumor types if new DRTs to relevant loci are developed.

A minority of melanocytic lesions cannot confidently be classified as benign or malignant on histopathological examination, causing diagnostic uncertainty. DNA copy number changes can be used to distinguish nevi from melanoma, although the use of FFPE tissue can pose technical challenges. DNA copy number assays, called duplex ratio tests, have been developed with duplex real-time PCR, using a simple method with potential for high throughput. Five duplex ratio test assays targeting loci with common DNA copy number changes in melanoma were designed and tested using DNA extracted from FFPE samples microdissected from melanoma, common nevi, benign tonsil (10 each), and two melanoma cell lines. The assays proved accurate when DNA extracted from fresh and FFPE melanoma cell lines were compared (intraclass correlation coefficient, 0.99) and gave precise results when repeated on DNA from FFPE tissue (intraclass correlation coefficient range, 0.90 to 0.96). In combination, duplex ratio test values from three of the assays distinguished between the nevi and melanomas with 100% sensitivity (95% CI, 69.1% to 100%) and 100% specificity (95% CI, 69.1% to 100%). Duplex ratio test assays have been shown to be accurate and precise and can distinguish melanomas from common nevi using DNA from FFPE tissue. Appropriately designed assays could have value in assessment of other cancers.