Application of 3-dimensional reflectance confocal microscopy: Melanocytic proliferations as three-dimensional models

Abstract

To the Editor: Melanoma on chronically sun-damaged skin (MSDS) is challenging to detect.1Sanchez M.I. Rabinovitz H.S. Oliverio M.C. Elgart G.W. Grichnik J.M. Confocal microscopy of challenging dermoscopic diagnoses.Arch Dermatol. 2012; 148: 1224Crossref PubMed Scopus (1) Google Scholar Reflectance confocal microscopy (RCM) increases diagnostic accuracy but relies upon interpretation of 2-dimensional (2D) horizontal sections. Our objective was to determine whether existing technology could be used to create 3-dimensional (3D) models, which would expand on data available for discriminating skin biology. Melanocytic cells have different morphologic appearances dependent on developmental stage, activation status, and pathogenic genetic and epigenetic alterations. Cell shape, therefore, can provide clues to a cell's behavior. We reconstructed individual melanoma cells from stacks of RCM images (Fig 1; Supplemental Fig 1, available via Mendeley at https://data.mendeley.com/datasets/7kx38d9h2v/3). Distinct morphologies could be identified, including spindle, spheroid, and heavily dendritic cell forms. These 3D reconstructions also allowed for modeling of the nuclear shapes (Fig 1, insets). Because image sizes are known, it is possible to calculate nuclear and cellular volumes, which may also increase diagnostic accuracy.2Chang C.K. Tsai C.C. Hsu W.Y. et al.Segmentation of nucleus and cytoplasm of a single cell in three-dimensional tomogram using optical coherence tomography [published correction appears in J Biomed Opt. 2017;22(3):39801].J Biomed Opt. 2017; 22: 36003Crossref PubMed Scopus (11) Google Scholar Monomorphism or pleomorphism of melanocytes may also provide clues to diagnosis. In some tumors, cells are relatively uniform, presenting as sheets of spindle-shaped cells, monomorphic spheroidal-shaped cells, or heavy mats of dendrites. However, other tumors display more variation with epithelioid, spindle-shaped, and dendritic morphologies all appearing in a 500-μm field (Supplemental Fig 2). Location is also important in diagnosis. The presence of melanocytic cells in the middle to upper epidermis (pagetoid spread) may suggest malignancy, whereas cells in the superficial dermis suggest invasion. Within the context of the 3D volume, we used renderings to show upper (epidermal) and lower (dermal) regions simultaneously (Supplemental Fig 3). This can be used not only for identification of pagetoid and invasive cellular locations but also for examining the extent of melanization of cells in different locations that may imply differences in maturation. When visualizing normal pigmented skin and some benign pigmented neoplasms with RCM, bright rims (edging) around the dermal papillae are visible as rings when viewed in the horizontal plane, primarily due to melanin-containing basal keratinocytes. By rendering the stack on the vertical axis, the normal arrangement of refractile cells along the papillae is apparent (Fig 2, Supplemental Fig 4). Epidermal thickness can be calculated, as well as the length of epidermal ridge and volume of dermal papillae in 3D. When MSDS is present, asymmetric pigmentation (partially edged or nonedged dermal papillae) may be noted around dermal papillae.3Serrano P.F.-C. Segurado-Miravalles G. González S. Reflectance confocal microscopy in oncological dermatology.in: Nouri K. Lasers in Dermatology and Medicine. Springer, 2018: 375-400Google Scholar This phenomenon may be due to nonuniform melanin transfer or melanoma cell population density differences. The dermoepidermal junction also frequently becomes flattened in malignant tumors. These phenomena may be best evaluated in 3D. The 3D modeling process currently has limitations. When manual segmentation techniques are used, the more complex models take hours to produce, limiting bedside use. The imaging area is limited to the stack dimensions (500 μm × 500 μm). RCM image stacks are anisotropic, thus the noncubic voxels require interpolation and resampling to render accurately in 3D space.4Trusk T.C. 3D reconstruction of confocal image data.in: Gray W. Price J.R.L. Basic Confocal Microscopy. Springer, Cham2018: 279-307Crossref Google Scholar In summary, 3D modeling of confocal images can provide supplementary information, beyond what is appreciable in 2D images, including melanocyte morphology, volume, pleomorphism, and location. 3D modeling also allows for detailed evaluation of the dermoepidermal contour, which may reflect the summation of keratinocytic, melanocytic, immunologic, and microenvironmental events.