Molecular testing: Multimarker assay demonstrates high accuracy in melanoma diagnosis

Key Points

San Francisco - A molecular test analyzing the intensity and distribution of expression of a handful of proteins in melanocytic lesions appears to provide a highly accurate method for discriminating between benign nevi and malignant melanoma.

Mohammed Kashani-Sabet, M.D., professor of dermatology and director of the Melanoma Center, University of California, San Francisco, and colleagues reported on the multimarker assay in a recently published paper (Proc Natl Acad Sci. USA 2009;106:6288-72). In initial testing of a diagnostic algorithm created using a training set of 118 benign nevi and 416 primary lesions, the molecular test achieved 91 percent sensitivity and 95 percent specificity.

Validation testing was then performed using four independent sets of tissues selected because they represent common diagnostic challenges (melanoma arising in a nevus, dysplastic nevi and Spitz nevi, misdiagnosed malignant melanomas).

Diagnostic method

The project took advantage of previous research conducted by Dr. Kashani-Sabet and colleagues in which they used gene expression profiling to identify genetic markers that might help in understanding the biologic behavior of melanoma.

In their earlier research, cDNA microarray analysis was used to profile the expression of more than 20,000 different genes in benign moles and melanomas representing various stages of tumor progression. The results demonstrated clear differences in genetic signatures between the lesion types.

"However, because performance of this assay requires fresh tissue - and considering that significant differences in expression were found for about 1,000 genes - it would be impractical to try to develop it as a tool that would be useful in clinical practice," he says.

The analysis developed is based on immunohistochemical protein staining that could be performed using paraffin-embedded tissue and focused on only five markers (ARPC2, FN1, RGS1, SPP1 and WNT2), which in the original microarray analysis were significantly overexpressed in the melanomas compared with benign moles.

The intensity of expression of the marker proteins was scored by a blinded dermatopathologist.

Study results

The results showed significantly greater expression of each of the protein markers in the malignant lesions compared with the benign nevi. However, the discriminatory performance of the test was enhanced when the data from all five markers was used, compared with from a single protein or any combination of less than five.

Separate scoring of protein expression in the top and bottom portions of the specimen was performed based on the finding that there were differences in the distribution of the markers between the benign and malignant lesions.

Whereas the five markers were homogenously expressed throughout the melanomas, in the benign nevi, marker expression was greatest in the most superficial portion of the lesion and decreased from top to bottom.

Digital imaging analysis was used to assess expression of a single protein, WNT2, and the results correlated strongly with the dermatopathologist's determination.

The University of California holds a patent on the protein analysis and has licensed the technology for commercial development to a biotech company that is now doing additional confirmatory work as it seeks Food and Drug Administration approval for marketing.

Disclosure: Dr. Kashani-Sabet has stock ownership in Melanoma Diagnostics.