Immunohistochemical and ultrastructural features of congenital melanocytic naevus cells support a stem-cell phenotype

Abstract

Background: Multiple congenital melanocytic naevi (CMN) in one individual are caused by somatic mosaicism for NRAS mutations; however, the lineage of the mutated cells remains uncertain.

Objectives: To test the hypothesis that CMN may be derived from cutaneous stem cells.

Methods: Sixty-six CMN samples from 44 patients were stained for immunohistochemical (IHC) markers of melanocytic differentiation (TYR, TRP1, TRP2, LEF1, MITF, cKit), pluripotency (nestin, fascin, CD133, CD20, CD34), monocyte/macrophage lineage (CD68, CD163, CD14), proliferation (Ki67) and MTOR/Wnt-signalling pathway activation (pS6, β-catenin). Semiquantitative scoring compared samples with naevus cell nesting (group 1) with those with only diffuse dermal infiltration (group 2). Transmission electron microscopy (TEM) was performed on 10 samples.

Results: A normal melanocyte population was seen overlying many dermal CMN. Group 1 samples were significantly more likely to express melanocytic differentiation markers than group 2, and expression decreased significantly with depth. Expression of these markers was correlated with each other, and with nestin and fascin. CD20 staining was positive in a substantial proportion and was stronger superficially. Expression of β-catenin and pS6 was almost universal. Some samples expressed monocyte/macrophage markers. TEM revealed variable naevus cell morphology, striking macromelanosomes, double cilia and microvilli.

Conclusions: Congenital melanocytic naevi development frequently coexists with normal overlying melanocyte development, leading us to hypothesize that in these cases CMN are likely to develop from a cell present in the skin independent of, or remaining after, normal melanocytic migration. IHC and TEM findings are compatible with CMN cells being of cutaneous stem-cell origin, capable of some degree of melanocytic differentiation superficially.

Figure 1

Staining for tyrosinase in two different samples; (a) ×20, scale bar 100 μm; (b) ×40, scale bar 50 μm; showing a normal melanocyte population along the basement membrane overlying dermal congenital melanocytic naevi.

Figure 2

Positive staining for melanocytic differentiation markers, (a) TRP1 (b) MITF (c) LEF1 and (d) cKit, all showing increased staining superficially and little or no staining deeply. Staining for these markers was generally reduced in samples without a nesting pattern. Staining for (e) nestin, (f) fascin, (g) β-catenin (cytoplasmic) and (h) pS6 showed less variability with depth of the lesional cells.

Figure 3

Congenital melanocytic naevi samples without a nesting pattern showed (a) CD20 staining in the majority (main picture ×4, scale bar 100 μm; inset from area within black square ×40, scale bar 50 μm); in a small subset (b) CD163 staining naevus cells and dermal dendritic cells (×20, scale bar 100 μm) and (c) CD14 staining naevus cells (×20, scale bar 100 μm).

Figure 4

Transmission electron micrographs of congenital melanocytic naevus cells; (a) naevus cell showing large nucleus with indentations, melanosomes in cytoplasm producing melanin, ×2500, scale bar 500 nm; (b) naevus cells in a nest encased in basal lamina (arrow), ×800, scale bar 2 μm; (c) variability in melanin production between neighbouring cells, ×5000, scale bar 1 μm; (d) inclusion body in nucleus (arrow), ×2000, scale bar 2 μm; (e) inclusion bodies in cytoplasm, and microvilli (arrow), ×1200, scale bar 2 μm; (f) macromelanosome complex, ×12 000, scale bar 500 nm; (g) double cilia seen as parallel lines (arrow), scale bar 500 nm; (h) cluster of naevus cells in macroscopically normal skin, ×3000, scale bar 2 μm.

Figure 5

(a) Perifollicular patterning of pigmentation at the borders of a single congenital melanocytic naevus (CMN) pre-resection, and (b) perifollicular regrowth after total macroscopic resection, suggestive of a hair follicle-associated reservoir of naevus cells; (c) palmoplantar CMNs are common in individuals with multiple CMNs, indicating that CMNs can also arise in areas devoid of hair follicles.