Malignant melanomas are the most aggressive skin cancers, with increasing morbidity in recent years. They include heterogeneous neoplasms characterized by different dermatoscopic, histological, and molecular profiles (1–3). A particular type of melanoma is the nodular variant, characterized by deep growth, frequent metastasis, and a high rate of genetic aberrations (4–6). Histologically, nodular melanomas (NMs) are composed of large, atypical spindle or epithelioid melanocytes, pleomorphic, sometimes organized in aggregates morphologically attributable to cerebroid nests of melanocytes (CNMs). They are mainly described in the peripheral portion of the tumors, where they assume an infiltrative profile or within the tumor mass. CNMs are round or oval in shape and are composed of aggregates of at least 5 undifferentiated atypical melanocytes, primarily devoid of pigmentation (7, 8). However, no attention has been paid to the count of nests detected in the neoplasm. No specific correlation has been investigated between their maximum concentration and clinical-pathological features.

In nodular melanoma, CNMs could be compared to poorly differentiated clusters (PDCs) of tumor cells. PDCs have been identified at the growing edge of the tumor in some types of epithelial cancers, including colorectal, gastric, breast, and endometrioid endometrial cancers (9–12). By definition, they are composed of ≥ 5 undifferentiated cells and are counted in the microscopic field under a × 20 objective lens (i.e., a microscopic field of 1 mm) (9, 13). In colorectal cancer, their highest number observed identifies the grade of malignancy: < 5 clusters for grade 1 (G1), 5 to 9 clusters for grade 2 (G2), and 10 or more clusters for grade 3 (G3) (14). The high number of PDCs is strongly associated with lymphatic vascular invasion and lymph node metastases, irrespective of the TNM stage (14–16). The correlation between the number of PDCs and the depth of infiltration of the submucosa in colorectal and gastric tumors is relevant (10, 14, 17). The unfavorable prognostic significance in terms of survival, demonstrated in studies with large case series of colorectal and gastric cancers, encourages to consider PDCs as possible tools in assessing the risk of lymph node involvement and progressive disease, regardless of pTNM stages and other histological features (10, 18). KRAS, NRAS and BRAF mutant colorectal carcinomas tend to form a high number of PDCs suggesting that the state of genomic instability may be a condition favoring their formation (19).

The aim of this preliminary study is to investigate the CNMs in a series of 25 NMs and to describe their association with their biological and histological characteristics.

The study included 25 NMs: 13 patients were males and 12 females, with a mean age of 67 years old (range 34 – 75 years). In detail, the mean diameter of the melanomas was 1.8 cm (range 0.9 – 2.3 cm), and tumor location included limbs (12 cases; 48%), chest-abdomen (8 cases; 32%), and other skin areas (5 cases; 20%). Breslow thickness was ≤ 1.0 mm in 13 cases (52%) (all pT1b) and > 1.0 mm in 12 (48%); Clark’s levels III, IV, and V were reached by 8 (32%), 15 (60%), and 2 (8%) tumors, respectively.

Epithelioid morphology of the tumor cells was observed in 16 cases (64%), and spindle cells in 9 of them (36%); 22 tumors (88%) showed > 5 mitosis x mm²; 6 masses (24%) were ulcerated. Lymph vascular invasion was observed in 12 cases (48%); TILs were present in 14 tumors (56%).

Eleven melanomas (44%) showed mutations. BRAF (V600E) mut was present in 6 cases (24%), NRAS (Q61R) mut in 4 cases (16%), and c-KIT (EXE11-G565E) mut in 1 case (4%).

CNMs were observed in the periphery of 15 melanomas (60%) ( Figure 1 ). Six of them (40%) were also detected within the tumor mass. The higher number of CNMs at 20x was seen mainly at the peripheral zone of the melanoma and, according to this, 17 melanomas (68%) were classified as G1 (0 – 4 CNMs), 5 (20%) as G2 (5 – 9 CNMs), and 3 (12%) as G3 (10 or more CNMs) ( Figures 2A–C ).

Histological and molecular features of the CNM+ and CNM- melanomas are listed in Table 1 . In detail, CNM+ tumors were mainly epithelioid in morphology (p = 0.041), showed more depth invasion (p = 0.042), showed more frequently lymph vascular invasion (p = 0.022) and a mutated status (p = 0.048); CNM+ melanomas were larger in diameter, although no statistical significance was reached (p = 0.068).

Histological and molecular features are listed in Table 2 , G1-CNM+, G2-CNM+, and G3-CNM+ melanomas, respectively. High-grade CNM+ melanomas (G3-CNM+) were more frequently ulcerated (p = 0.025), and BRAF (V600E) mutated (p = 0.028). Although statistical significance was not reached, they exhibited predominantly epithelioid cell morphology, and were lymph vascular invasive.

NM represents a clinically aggressive histologic variant characterized by atypical pleomorphic melanocytic cells with large cytoplasm, spindle or epithelioid shape, and prominent nucleoli (3, 20, 21). In many tumors in the periphery, CNMs can be detected, which detach from the main mass, taking on an infiltrative appearance. CNMs are readily detectable in slides stained with H&E and show a morphology similar to the aggregates of PDCs observed in colorectal carcinoma (9). Although CNMs are described in NMs, no studies currently consider their histopathologic or prognostic significance. Concerning this, we examined the histological slides of a selected group of NMs and demonstrated the presence of CNMs at the periphery of 15 (60%) of them and a number ≥ 10 nests at 20x field in 3 cases (12%). We showed that CNMs were significantly associated with lymphatic vascular invasion, and higher Clark level, suggesting a possible role in tumor growth. This would be responsible for vascular invasion and, thus, metastasis, typical of their more aggressive clinical behavior (20, 22–28). Therefore, CNMs represent the active growth front of these melanomas, typically observed in epithelial malignancies. In support of this, it is interesting to report that CNMs were significantly associated with the epithelioid phenotype of the melanoma from which they originated. As shown in Figure 3 , where we compare PDCs and CNMs, we hypothesize that NMs with epithelioid morphology have an aggressive growth attitude, probably comparable to poorly differentiated epithelial neoplasms with active neoplasm “buds” (17). Indeed, in our experience reported here, CNM+ melanomas reach Clark’s IV and V levels of infiltration and are clinically characterized by recurrence and metastasis (p = 0.011; Table 2 ). This aspect of invasiveness has also been similarly demonstrated in colorectal cancers with a high number of PDCs in tumors deeply infiltrating the intestinal wall (pT3 and pT4 tumors) (15, 16, 29).

CNMs are more abundant at the periphery of melanomas than those detectable within masses, which are rarer to identify. As surmised for colorectal PDCs (30, 31), this could indicate that their origin derives from the epithelio-stroma interaction, which belongs to the mechanisms that drive cell growth and proliferation (30). This aspect is typically observed in epithelial tumors (32) and therefore now acceptable as a possible explanation to the formation of PDCs (14). It must, however, be transferred with caution in the context of NMs with CNMs since the origin is neuroectodermal and for that reason melanoma cells may not be subject to epithelial-mesenchymal transition (EMT)-mediated effects (33). On the other hand, it is also known that in the dermal invasive phase, tumor melanocytes acquire molecular changes in cell-cell adhesion proteins, including the downregulation of the junctional protein E-cadherin (33–35) and that this process becomes particularly evident during the transition from radial to vertical growth phase (36). On the other hand, it should be mentioned that nonepithelial tumors, including melanoma, can acquire mesenchymal-like properties (37). However, it is still not entirely clear how transcription factors that induce EMT drive the growth and progression of nonepithelial tumors, such as melanoma (37, 38).

In recent years, it has been identified that genetic factors, including activating mutations in the BRAF and NRAS oncogenes, contribute to melanoma initiation, promoting its growth and metastasis, as well as the transition of melanoma cells into different epithelial and mesenchymal states (39). Our study reported that BRAF (V600E) mutated NMs have a higher number of CNMs at the periphery of the mass, suggesting that the accumulation of genetic mutations in tumor cells could promote the creation of clones of cells that can aggregate and migrate by invading the dermis. Studies on PDCs and colorectal cancer report that a high number of PDCs in colon cancer are associated with the V600E mutation in the BRAF gene, although without statistical significance due to the limited number of cases collected (14, 19). BRAF is a Ras-activated serine/threonine protein kinase that participates in the MAP kinase/ERK signaling pathway (40), mutated in approximately 50% of melanomas [Catalogue of Somatic Mutations in Cancer (COSMIC) at http://www.sanger.ac.uk/cosmic] (41). BRAF (V600E) mut has been implicated in melanoma progression, senescence evasion, apoptosis, uncontrolled replication potential, and angiogenesis, resulting in tissue invasion and metastasis (42). The interaction between melanocytic tumor cells and the surrounding microenvironment, consisting of different extracellular matrix components and growth factors, and the induction of EMT in melanoma are influenced by common mutations and/or deregulated expression of BRAF, NRAS, and PTEN, which appear to act synergistically with each other and with different microenvironmental factors (43). The microenvironment can integrate aberrant genetic changes such as mutations in BRAF to promote melanomagenesis and support an invasive cell phenotype. Therefore, BRAF mutation could promote the genesis of tumor melanocyte clones that would take on the biomolecular characteristics required to generate CNMs (40, 44).

Our study has some limitations: first, we have a “small” series of NMs, and second, molecular mutations were detected in only 11 out of 25 cases, but this is due to the difficulty in enrolling cases with CNMs as well as the fact that this is a pilot study, so the results should be considered preliminary.

In conclusion, CNMs represent a promising additional unfavorable histologic feature of NMs with epithelioid morphology. Because CNMs+ melanomas appear to be associated with the BRAF V600E mutation, the presence of CNMs could have cancer predictive significance for identifying a subgroup of patients who might benefit from specific biologic drugs. However, further studies are needed to confirm this hypothesis.

The original contributions presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding author.

The study was approved by the Ethical Review Committee of the Modena University Hospital (protocol number CE 289\13) and conducted according to the Helsinki Declaration. Written informed consent was obtained from each patient enrolled. The studies were conducted in accordance with the local legislation and institutional requirements. Written informed consent for participation in this study was provided by the participants’ legal guardians/next of kin.

SC: Writing – original draft. AM: Writing – original draft, Writing – review & editing. LB: Conceptualization, Formal analysis, Writing – original draft, Writing – review & editing. MM: Visualization, Writing – review & editing. FF: Visualization, Writing – review & editing. PP: Visualization, Writing – review & editing. VA: Visualization, Writing – review & editing. GC: Visualization, Writing – review & editing. TS: Writing – review & editing. GP: Visualization, Writing – review & editing. LRB: Conceptualization, Formal analysis, Writing – original draft, Writing – review & editing.