Congenital Melanocytic Nevi (CMN) defined by their presence at birth or in the first few weeks of life originate from benign melanocytic proliferations of the neural crest. The clinical features of CMN are various such as body site, a more diverse spectrum of colors, and surface topographical changes. Potential complications from CMN include melanoma development and central nervous system involvement termed Neurocutaneous Melanosis (NCM).

The classification of CMN has historically been defined by size alone. Recent recommendations were published based on expert consensus, utilizing Predicted Adult Size (PAS) categories, the number of accompanying CMN, anatomic location, and several topographic descriptors. A small CMN is defined as less than 1.5 cm PAS and medium up to 20 cm PAS (M1: 1.5–10 cm, M2: >10–20 cm). Large and giant CMN are classified as those CMN with PAS between 20 and 40 cm (L1: >20–30 cm, L2: >30–40 cm) and greater than 40 cm (G1: >40–60 cm, G2: >60 cm), respectively.1

Small and medium CMN are reported approximately in 1 in 100 births.2 while Large and giant CMN has an estimated prevalence of 1 in 20,000 to 1 in 50,00 births and 1 in 200,000 to 1 in 500,000 births respectively.3–5

CMN harbored predominantly a post-zygotic somatic NRAS or BRAF mutation. Nevertheless, different mutations usually depend on nevus size. In the vast majority of cases, small and medium CMN is more frequently noted to have a BRAF V600E mutation, while large/giant counterparts more frequently harbor NRAS (Q61) mutations.6–8 CMN are usually benign melanocytic neoplasms, but sometimes it is hard to differentiate CMN from melanoma, especially at an early stage of melanoma. Therefore, biopsies or surgery are needed to make a further pathologic examination. The BRAF V600E mutation is the most common genetic driver in melanoma and encodes a serine/threonine kinase in the RAS/Mitogen-Activated Protein Kinase Pathway (MAPK).9–12

Remarkably, more than 80% of melanocytic nevi harbor BRAF mutations,10,13 including the melanocytic nevi that act as precursors for melanoma.14

In melanoma, the presence of the BRAF V600E mutation correlates with certain histopathologic features, including intraepidermal upward scatter of melanocytes, nest formation of intraepidermal melanocytes, thickening of the epidermis, and larger tumor cells.15 Similarly, melanocytic nevi with a BRAF V600E mutation are associated with a globular dermoscopic pattern and a predominantly dermal histologic growth pattern.13,16 These results suggest that the BRAF V600E mutation may affect the characteristics and behavior of nevi.

Mutations in BRAF (v-raf murine sarcoma viral oncogene homologue B) have been detected with high frequency in CMN, especially in small and medium CMN.17 However, as far as we know, there are no relevant reports on the effect of BRAF V600E gene mutation in CMN on proliferation activity and histopathology. As such, the study of the biology of CMN may further inform the understanding of pathways leading to melanoma.

In this study, the primary objective was to determine the proliferation activity (by Ki67) and histopathological pattern in CMN with BRAF V600E gene mutation.

This study was approved by the institutional review board of the Capital Institute of Pediatrics (SHERLL2020054) and was carried out in accordance with the Declaration of Helsinki.

Laser confocal fluorescence microscopy (LCFM)

If there were too many melanin particles in the specimen, which affected the judgment of the results, the laser confocal fluorescence microscope was used to further clarify the diagnosis. Sections were deparaffinized in xylene and rehydrated with 100%, 95%, 85% and 75% ethanol for 5 min respectively and rinsed with phosphate-buffered saline for 5 min for 3 times. After microwave heat-induced epitope retrieval for 15 min and blocking in 5% normal goat serum for 1 hour at room temperature, sections were stained with monoclonal mouse anti-human Melanin A and rabbit antihuman Ki-67 antibody (Abcam, Cambridge, MA, USA) in 5% bovine serum albumin/Tris-buffered saline. For detection, AlexaFluor 488-labeled goat anti-mouse and AlexaFluor 595-labeled goat anti-Rabbit antibodies (Abcam, Cambridge, MA, USA) and DAPI were used. For colocalization analysis, cells were simultaneously labeled with anti-Melanin A mouse antibodies and anti-Ki-67 rabbit antibodies. and detected with AlexaFluor 594-labeled goat anti-mouse or AlexaFluor 488-labeled goat anti-rabbit antibodies. The procedure was performed manually. Positive and negative controls were included with each run of samples. The percentage of positive cells was counted under a high-power view (Fig. 1).

Figure 1.

Histomorphologic features of CMN. Congenital growth pattern in nevi involving adnexal epithelium and wrapping of nevus cells around vessels and splaying of melanocytes in-between collagen bundles (A‒B). But the nevi with BRAF V600E mutation (A) would spread into deep dermis even deeper than wild type nevi (B). (A‒B, Hematoxylin & eosin, ×40).

(1.09MB).

Data on the 12 matched pairs including patients (n = 24) and nevi (n = 24) are shown in Table 1. Characteristics of CMN of mutation group and controls. The mutant and control groups were closely matched in age, sex, and location. There were no statistically significant differences in age, sex, history of melanoma, and history of non-melanoma skin cancer between the mutant and control groups.

There were 21 compound nevi in this study (21 of 24, 87.5%). Nevertheless, there was only one intradermal nevus in the mutation group (8.3%), while two were in the control group (16.6%).

Ki67 staining was restricted to the nucleus of positive cells in a distinct granular pattern of staining and was often inhomogeneous. Occasionally the nucleolar region was stained prominently. No cytoplasmic staining was observed. More frequent staining was observed at the dermal-epidermal junction (Figs. 2 and 3).

Figure 2.

Ki67 immunohistochemistry of CMN. Ki67 staining was restricted to the nucleus of positive cells in a distinct granular pattern of staining and more frequently observed at the dermal-epidermal junction (A‒B, ×100).

(0.76MB).

Figure 3.

The brown stained nuclei indicate Ki67 positive expression (A, ×400). Confocal laser fluorescence microscopy can easily eliminate the interference of melanin granules. Cells with red fluorescence in the center and yellow fluorescence around are Ki67 positive cells (B ×600).

(0.66MB).

Table 2 shows the proportion of cells positive for the Ki67 staining in each nevi pair. Statistically significant differences were detected between the mutation group and the control (p = 0.041).

The differences in the depth of nevus cell involvement and the number of nevus cell nests between the mutant group and the control group were statistically significant, with p-values of 0.002 and 0.007, respectively, while the difference in the size of cell nests between the two groups was not statistically significant (Table 2).

Among the cases, the correlation between the number of the nevus cell nests and the Ki67 positivity rate is shown in Fig. 4. The number of the nevus cell nests (Linear regression analysis, Spearman’s correlation coefficients: coefficient = 0.603; p = 0.001; Fig. 1) was strongly correlated with the proportion of cells positive for Ki-67 staining. However, Ki-67 staining had no significant correlation with nevus cell depth, nevus cell nest size, and nevus size, and the correlation coefficients were 0.11, 0.15 and 0.01, respectively.

Figure 4.

Correlation between Ki-67 and nest numbers.

(0.19MB).

BRAF immunohistochemical staining was performed in 10 cases in a blinded manner (i.e., without knowledge of the results of the gene sequencing before analysis of the immunohistochemical staining data). All 7 nevi with negative immunohistochemical staining for BRAF V600E were negative for BRAF mutation, and all 3 nevi with positive immunohistochemical staining for BRAF V600E had BRAF V600E gene mutation. No other recurrent somatic mutations were identified.

At present, there are only a few literature reports about the association of gene mutation with the pathology and proliferation activity of CMN.13,19,20 These reports were holistic studies and did not distinguish between congenital and acquired nevi. To our knowledge, this is the first study to demonstrate the relationship between BRAF mutation on the proliferation activity and histopathology of CMN.

According to microscopic histologic characteristics, CMN may be junctional, compound or exclusively intradermal. The histologic characteristics are related primarily to the size of the nevus.21 Nearly all nevi included in this study were compound (21 of 24, 87.5%) and the histopathological features were consistent with the congenital pattern, such as symmetry, good circumscription, uniform nests in the epidermis equidistant from each other, nests of melanocytes and individual melanocytes in the dermis that mature with their descent, wrapping of nevus cells around vessels and adnexal structures, and splaying of melanocytes in-between collagen bundles.22 It's worth noting that the difference in nevus depth was significant between the two pair-matched groups. The nevus cells in the BRAF mutation group extend deeper than the non-mutation group. This phenomenon may be caused by the enhanced proliferative activity of nevus cells after the BRAF gene mutation. The depth of nevus infiltration was also positively correlated with the size of the nevus.21 By reason of the foregoing, the authors can draw a preliminary conclusion that the depth of nevus infiltration is not only related to the size of nevus, but also related to the mutation of BRAF V600E gene in CMN. Additionally, the findings of this study expand the knowledge of genotype-phenotype correlation in CMN.

The monoclonal antibody of Ki-67 has been widely used in histopathology and shown to be of particular value for tumor grading and to correlate with patients’ survival in many human malignancies.23 But data on the expression of proliferative activity marker Ki67 in CMN are limited. Soyer included benign melanocytic nevi in his study and found significant differences in Ki-67 antigen expression between nevi, primary and metastatic malignant melanoma.24

BRAF V600E is observed in nevi with a high frequency10 and is a driver mutation in melanoma pathogenesis.25 BRAF V600E transiently activates nevi melanocytes proliferation and induces p16 that in response halts proliferating melanocytes.26 But the arrest is reversible since some nevi ultimately transform into skin melanomas.27 There is a dynamic balance between hyperplasia and inhibition of nevi, and if this balance is broken, it is likely to develop into malignant melanoma.

In the present study, the authors find that there is a statistically significant difference in proliferation activity between nevi with BRAF V600E mutation and the control group. Hence, with the BRAF V600E mutation, CMN can have more proliferative activity and maybe more susceptible to other irritant factors, such as sun exposure and friction, and would eventually progress to malignant melanoma.

As far as the authors know, we have demonstrated that the proliferation activity of CMN is positively correlated with the number of nevus cell nests but not with the nest size. Hence, the nest number may be an index of proliferative activity and could be used in a future study.

The above research is preliminary, and the results will have to be validated in a larger scale of study. Additional studies of nevi promise to inform understanding of the mechanisms of nevogenesis and the transition from benign neoplasm to malignancy.

Our study demonstrates that BRAF V600E gene mutation can enhance proliferative activity and can affect the histopathological features of CMN in children. While on the other hand, the histopathological features of a CMN can provide relevant information on its molecular background, including the presence of BRAF V600E gene mutation, which is the most common driver of melanocytic tumors and the most common therapeutically targetable alteration in melanoma. A greater understanding of the genotype-phenotype correlation in CMN may facilitate a more accurate diagnosis and more proper management of these exceedingly common human melanocytic tumors.

This work was supported by the Research Foundation of the Capital Institute of Pediatrics(project number: LCPY-2021-04).

Jianyou Chen: Approval of the final version of the manuscript; design and planning of the study; drafting and editing of the manuscript; collection, analysis, and interpretation of data; effective participation in research orientation; critical review of the literature; critical review of the manuscript.

Gaolei Zhang: Approval of the final version of the manuscript; design and planning of the study; drafting and editing of the manuscript; critical review of the literature.

Xiaoyan Liu: Approval of the final version of the manuscript; collection, analysis, and interpretation of data; effective participation in research orientation.

Ping Tu: Statistical analysis; approval of the final version of the manuscript; collection, analysis, and interpretation of data; effective participation in research orientation; critical review of the literature; critical review of the manuscript.

None declared.