800 Genomic detection of melanoma, W. Wachsman et al. Results Use of epidermal genetic information retrieval-harvested RNA to identify genes differentially expressed between melanoma, naevi and normal skin In this study, we sought to address whether the EGIR method can be used to discern between melanoma, naevi and normal skin. We harvested 29 melanomas (in situ and invasive), 68 naevi (benign and atypical) and 15 normal skin specimens from which RNA was isolated, ampliﬁed and proﬁled on the Affymetrix U133 plus 2.0 GeneChip. ANOVA analysis (P < 0Æ001, Westfall and Young permutation multiple testing correction,24 FDR q < 0Æ05) of the resulting microarray data identiﬁed 312 differentially expressed genes (the annotated list of genes is provided in Table S1; see Supporting Information). Hierarchical clustering analysis of these 312 genes showed two major branches in the heat map (Fig. 1). The branch on the left side of the heat map contained all 29 melanomas and 13 of the naevi and was clearly separated from the right branch that contained the bulk of the naevi as well as all of the normal skin specimens. Most of the 13 naevi that grouped together with melanomas were Clark (also known as ‘dysplas- tic’) naevi with mild to severe cytological atypia, and as such bear some morphological resemblance to melanoma. Of note is that normal skin specimens could not be separated from naevi based on the expression of these 312 genes, suggesting that the RNA in the stratum corneum over these lesions is similar. Most of the naevi that grouped together with normal skin were nondysplastic, compound, intradermal or junctional naevi. Taken together, these ﬁndings demonstrate that expres- sion proﬁling of EGIR-harvested RNA from stratum corneum can be used to identify genes differentially expressed between melanoma, naevi and normal skin. Biological functions of the 312 genes related to melanoma The biological functions and molecular networks of the 312 genes were assessed by IPA and, interestingly, genes upregulat- ed in the heat map cluster containing melanoma were found to play a role in melanocyte development and pigmentation signalling as well as skin development, cellular development and cancer (Table 2). Some of the genes upregulated in these EGIR-harvested stratum corneum specimens overlying melanoma are shown in Figure 2 including KIT, tyrosinase (TYR), tyrosinase-related protein 1 (TYRP1), dopachrome tau- tomerase (DCT), SOX10 and PAX3 – all of which are involved in melanocyte development and pigmentation. In addition, other genes known to be expressed in melanocytes, including MLANA, SILV, EDNRB and melanophilin, were also detected in these EGIR specimens overlying melanoma. Some of the genes upregulated in the heat map cluster containing naevi and nor- mal skin are involved in cellular growth and proliferation, embryonic development, and skeletal and muscular system development and function. These ﬁndings support the hypoth- esis that the EGIR method can be used to distinguish mela- noma from naevi and normal skin. Further, these observations suggest that the gene expression proﬁle of mRNA found in the stratum corneum overlying a pigmented lesion is altered, either directly or indirectly, by the presence of melanoma. Identiﬁcation of a 17-gene classiﬁer to distinguish melanoma from naevi Having demonstrated that EGIR tape-stripped specimens can be used to detect melanoma, we next asked whether we could develop an approach to predict the risk that a pigmented lesion contains a melanoma. Our strategy was to develop a Fig 1. Hierarchical clustering analysis of differentially expressed genes among melanomas, naevi and normal skin. These 312 genes determined from microarray analysis of epidermal genetic information retrieval specimens differentiate melanoma from atypical naevi and normal skin (P < 0Æ001, false discovery rate q < 0Æ05). BJD 2011 The Authors 2011 British Association of Dermatologists 2011 164, pp797–806