In-situ-topoproteome analysis of cutaneous lymphomas: perspectives of assistance for dermatohistologic diagnostics by Multi Epitope Ligand Cartography (MELC)

Immunophenotyping is essential for diagnostics of cutaneous lymphomas. In this regard we present a skin tissue-adapted application platform of MELC technology. This topoproteome analysis allows the subcellular colocalization of at least n = 100 epitopes in situ. For this purpose the specimen is processed by a Toponome Imaging Cycler for a n-fold repetition of the following cycle: 1) staining with a fluorophore-labeld antibody, 2) fluorescence-imaging, and 3) photobleaching. Overlay and binarization of fluorescence images lead to combinatorial molecular phenotypes (CMP), which relate to a pixel or microtopographic unit (450 x 450 nm2, 20x objective). Skin biopsies were derived from patients with mycosis fungoides (patch/plaque lesions), psoriasis, atopic eczema and from healthy skin donors. In orientation to the WHO-EORTC-classification of cutaneous lymphomas a MELC-library of 23 markers was established. According to an inaugurative detailed procedure the CMP frequency was determined in a normalization to 100 microm horizontal skin width. By a TopoMiner strategy mycosis fungoides could be separated from the other states with a maximum of significance (p < or = 0.03) by at least 10-fold overexpression of the following tumor cell-representative CMP-motif: CD3+/CD4+/CD1a-/CD7-/CD8-/CD45R0+/CD45RA-/CD11a+. The skin tissue-adapted MELC-application-platform extends substantially conventional lymphoma diagnostics by an unprecedented dimension of in-situ-analysis of marker combinatorics including its exact quantification and visualization.