804 Genomic detection of melanoma, W. Wachsman et al. Fig 3. Hierarchical clustering of melanomas and naevi using the 17-gene classiﬁer. Shown are data from the training set of 37 melanomas and 37 naevi. The specimen denoted by the arrow was called a Clark naevus on initial pathological review, but was deemed a melanoma by the 17-gene classiﬁer. The presence of invasive melanoma was detected in this biopsy by both the primary and central dermatopathologists upon re-review of the serially sectioned specimen. melanoma from basal cell carcinoma, solar lentigo and normal skin. The 17-gene classiﬁer falsely identiﬁed 13 naevi as mela- noma, raising the question as to why these were misclassiﬁed. One possibility is due to a sampling error by histopathological examination of a limited number of sections from the biopsied specimen. This is clearly the case for one of the specimens (denoted by an arrow in Fig. 3), which when serial sectioned and re-reviewed was found to harbour a melanoma. This had been missed during the initial histopathological reading due to the fact that serial sectioning of the biopsied lesion is not rou- tinely performed per standard of care. Another possibility is that EGIR-based genomic assay can detect molecular changes prior to the development of morphological abnormalities in mela- noma cells. A third possibility is that these false-positive naevi exhibited pagetoid spread of melanocytes. After re-reviewing the tissue biopsy specimens we could ﬁnd little, if any, evidence of pagetoid spreading in these false-positive naevi. If these results can be conﬁrmed, it would suggest that the EGIR-based genomic assay may be a more sensitive means to detect mela- noma than the standard histopathological review. Tape stripping removes mainly the stratum corneum, the layer of the skin that does not contain nuclei. The pigment- producing cell, the melanocyte, however, resides in the basal cell layer of the epidermis. Thus, it is surprising that one can isolate mRNA, indicative of this neoplasm, from the skin’s horny layer. Several observations may explain this ﬁnding. It is known that the distal portion of the melanocyte’s dendritic processes are actively phagocytosed by keratinocytes and the melanin contained in these processes is then dispersed throughout the keratinocyte layers, eventually arriving in the stratum corneum.34 This appears to be the mechanism by which pigment moves to the upper ⁄ outermost layers of the integument from the melanocyte that produces the melanin. Conceivably, the mRNA synthesized by the melanocyte also moves upwards in the epidermis during this process. The keratinocyte may also phagocytose the dendritic processes of Langerhans cells. This would explain why mRNA indicative of an allergic reaction, thought to be a function of the Langer- hans cells resident in the suprabasal cell layer, can also be found in the stratum corneum.21 In addition, documentation of the mRNA indicative of an abnormal skin reaction at the skin’s surface may be explained by the recent observation that the dendritic processes of Langerhans cells penetrate through the tight junctions of the keratinocytes and exit the skin just beneath the stratum corneum where the RNA could be picked up by the tape stripping.35 Another possibility for our ﬁndings is the phenomenon of cell–cell crosstalk (see below). In this scenario the malignant melanocyte in the epidermis would activate the keratinocytes adjacent to it to generate melanocytic mRNAs, and this process would continue until the abnormal expression of mRNAs would manifest itself in the uppermost layer of the stratum granulosum when the nucleus becomes extruded. From there, the mRNAs indicative of a melanoma could be detected by tape stripping. Alternatively, the RNA from malignant melanocytes could be sampled by EGIR if the malignant cell is present in the stra- tum corneum or if tape stripping extends well below into the stratum granulosum. The former is unlikely because it is thought to be a rare event and therefore not capable of gener- ating a signal strong enough to overcome the surrounding noise of corniﬁed keratinocytes. The latter also would not provide a clear explanation, as histopathological review of specimens typically did not show that tape stripping resulted in penetration beyond the stratum corneum layer. However, it is possible that dendritic processes from the malignant melanocytes could extend into the stratum corneum that might be sampled during the process of EGIR. Recent ﬁndings on the genetics of melanoma have shown distinct subsets of the disease in which speciﬁc genomic aber- rations have been associated with histological subtypes of BJD 2011 The Authors 2011 British Association of Dermatologists 2011 164, pp797–806