Development and Validation of a Chromogenic RNA In Situ Hybridization Assay for Diagnosis of Atypical Melanocytic Nevi and Malignant Melanoma
Catharine L Kauffman, David Stout, Xingyong Wu, Doru Alexandrescu, Yuling Luo, Xiao-Jun Ma, Paul Shitabata. Georgetown Dermpath, Washington, DC; Advanced Cell Diagnostics, Hayward, CA; Dermatopathology Institute, Torrance, CA
Background: Accurately distinguishing benign melanocytic lesions from malignant melanoma remains one of the most difficult challenges in diagnostic pathology. In recent years, a 4-probe fluorescent in situ hybridization (FISH) assay assessing gross genomic aberrations has been developed, but its clinical adoption has been limited due to its focus on Spitz nevi as well as its requirement of specialized equipment.
Design: Archival formalin fixed paraffin embedded specimens of melanocytic skin lesions that had been diagnosed by three experienced dermatopathologists were retrieved from two institutions with institutional review board approval. We evaluated 23 genes identified from microarray analysis and the literature in 149 training samples (92 melanomas and 57 nevi) to develop a multi-gene RNA ISH assay for classification of benign nevi and melanomas. The final assay was validated in an independent cohort of 171 cases consisting of a broad spectrum of nevi (n=116) and melanomas (n=55).
Results: We identified three melanoma-specific markers (PHACTR1, SPP1 and PRAME) and three reference genes (MLANA, S100A6 and S100B). Samples with positive staining for at least one of the three melanoma-specific markers were classified as malignant, which had a sensitivity of 97% and a specificity of 93% in the training set. Two (22%) of 9 severely atypical nevi in the training set were positive for at least one of the three melanoma markers. In the validation set, the same assay and scoring algorithm correctly classified 132 (91%) of the 145 unambiguous benign nevi and melanomas (89% sensitivity, 95% CI 78%-96%; 92% specificity, 95% CI 85%-97%). Eight (31%) of 26 severely atypical nevi, compared to 2 (4%) of 45 mild and moderate atypia specimens, stained positively for at least one of the 3 melanoma markers (p < 0.01). A higher number of positive melanoma markers in melanoma cells was significantly associated with a higher tumor stage (p < 0.01) and Clark level (p=0.05).
Conclusions: This slide-based assay performs with real-world diagnostic specimens of melanocytic lesions, and the results can be viewed under a standard light microscope without special equipment. It may serve as a powerful molecular adjunct in the diagnosis of early stage melanoma, especially in the setting of severely atypical nevi. These melanoma markers may also be prognostic, as evidenced by their correlation with established prognostic factors.
Monday, March 4, 2013 9:15 AM
Proffered Papers: Section F, Monday Morning