In vivo multimodal optical imaging of dermoscopic equivocal melanocytic skin lesions

Abstract

There is a wide range of equivocal melanocytic lesions that can be clinically and dermoscopically indistinguishable from early melanoma. In the present work, we assessed the possibilities of combined using of multiphoton microscopy (MPM) and optical coherence angiography (OCA) for differential diagnosis of the equivocal melanocytic lesions. Clinical and dermoscopic examinations of 60 melanocytic lesions revealed 10 benign lesions and 32 melanomas, while 18 lesions remained difficult to diagnose. Histopathological analysis of these lesions revealed 4 intradermal, 3 compound and 3 junctional nevi in the "benign" group, 7 superficial spreading, 14 lentigo maligna and 11 nodular melanomas in the "melanoma" group and 2 lentigo simplex, 4 dysplastic nevi, 6 melanomas in situ, 4 invasive lentigo melanomas and 2 invasive superficial spreading melanomas in the "equivocal" group. On the basis of MPM, a multiphoton microscopy score (MPMS) has been developed for quantitative assessment of melanoma features at the cellular level, that showed lower score for benign lesions compare with malignant ones. OCA revealed that the invasive melanoma has a higher vessel density and thicker blood vessels than melanoma in situ and benign lesions. Discriminant functions analysis of MPM and OCA data allowed to differentiate correctly between all equivocal melanocytic lesions. Therefore, we demonstrate, for the first time, that a combined use of MPM and OCA has the potential to improve early diagnosis of melanoma.

Figure 1

MPM of benign melanocytic lesions and melanoma. Representative MPM images of junctional (a–c), compound (e–g), intradermal (i–k) nevi and melanomas (m–o) at various depths and corresponding histopathological images (d,h,l,p). Stratum granulosum (a,e,i,m), stratum spinosum (b,f,j,n) and dermal–epidermal junction (c,g,k,o). The arrows show nevus cell nests (in c,g) and polymorphic cells (in m), pagetoid cells (in n) and dendritic structure (in o). Scale bar is 100 µm for all images. In MPM images, cellular autofluorescence (red) and SHG signal from collagen (green) are shown.

Figure 2

The malignant features revealed by MPM and OCA and confirmed by histopathology. The arrows show polymorphic cells (in a,d), dendritic structures (in b,e), pagetoid cells (in c,f), nests (in g,j), non-edged papilla (in h,k), nonvisible papilla (in i,l). In OCA images of vessel network the arrows show clusters of curved vessels with a diameter of ≤ 15 μm (in m,o) and clusters of irregularly distributed linear vessels with a diameter of ≥ 50 μm (in n). Scale bars are 100 µm for MPM and 1 mm for OCA.

Figure 3

MPM features of benign lesions in dermoscopic equivocal group. In lentigo simplex (a,b), bright nucleated cells in spinous layer (a) and brain-like structure in dermal–epidermal junction (b) are shown by the arrows. In dysplastic nevi (c,d), bright pagetoid cells (c) in spinous layer and atypical cells in rate-ridges (d) are shown by the arrows. Autofluorescence from the cells (red) and SHG signal from collagen (green) are shown. Imaging depth is marked on the images. Scale bar is 100 µm, applicable to all images.

Figure 4

MPM features of melanoma in dermoscopic equivocal group. In melanoma in situ, large pagetoid cells in spinous layer (arrow, a) and non-edged papillae (arrow, b) are marked. In lentigo maligna melanoma, large pleomorphic cells in spinous layer (arrow, c) and melanophages below dermal–epidermal junction (arrow, d) are marked. In superficial spreading melanoma, nest of atypical cells (arrow, e) and round pagetoid cells (arrow, f) in the epidermis are marked. Autofluorescence from the cells (red) and SHG signal from collagen (green) are shown. Imaging depth is marked on the images. Scale bar is 100 µm, applicable to all images.

Figure 5

The quantitative analysis of malignant features of dermoscopic equivocal lesions revealed by MPM and OCA. The MPMS (a), total length of small (≤ 15 μm) and large caliber vessels (≥ 50 μm) (b) and vessel density (c) for benign melanocytic lesions, melanoma in situ (MIS) and invasive melanoma. Whiskers show the mean and 95% confidence interval. Rhombus, squares, cycles and triangles display the measurements for individual patients. *, p < 0.05.

Figure 6

Representative OCA images of benign and malignant lesions from dermoscopic equivocal group. Vessel networks in lentigo simplex (a), dysplastic nevi (b), melanoma in situ (c), lentigo maligna melanoma (d), and superficial spreading melanoma (e). Scale bar is 1 mm, applicable to all images.

Figure 7

Discriminant function analysis of malignant features revealed in dermoscopic equivocal lesions. (a) Scatterplot represents discriminant functions (DF) distribution for benign lesions (blue cycle), melanoma in situ (red square) and invasive melanoma (green rhombus). (b) The standardized coefficients of discriminant functions DF 1 and DF 2. (c) Classification matrix. (d) Independent contributions to the prediction.