Received Date : 02-Dec-2013 Accepted Date : 27-Feb-2014 Article type : Viewpoint

"How, and from which cell sources, do nevi really develop?" James M. Grichnik,123 Andrew L. Ross,1 Samantha L. Schneider ,1 Margaret I. Sanchez,12 Mark S. Eller,2 Konstantinos E. Hatzistergos3
1

Department of Dermatology and Cutaneous Surgery Sylvester Comprehensive Cancer Center Interdisciplinary Stem Cell Institute, University of Miami Miller School of Medicine, Miami, Florida,

USA.

Corresponding author: James M Grichnik MD PhD Professor and Chief, Frankel Family Division of Melanocytic Tumors, Department of Dermatology and Cutaneous Surgery Director, Melanoma Program, Sylvester Comprehensive Cancer Center Member, Interdisciplinary Stem Cell Institute Miller School of Medicine, University of Miami Room 912, BRB 1501 NW 10th Ave Miami, FL 33136 Email: grichnik@miami.edu Office 305-243-6045 This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1111/exd.12363 This article is protected by copyright. All rights reserved.

Abstract Melanocytic neoplasms are a diverse group of benign and malignant tumors with variable clinical features. While some models still promote the epidermal melanocyte as the origin of melanocytic neoplasms, clinical findings are inconsistent with this theory for the majority of tumors. Despite advances in nevus and melanoma biology, the location and differentiation status of the cell of origin remains undefined. Germ line genetics, biological state and cellular location of the mutated cell, as well as local environmental factors all likely play a role in the development of melanocytic neoplasms. Herein, we will review potential models for melanocytic neoplasia and discuss research challenges and opportunities.

Keywords: melanoblast, melanocytic stem cell, melanoma stem cell, nevogenesis, melanomagenesis

Introduction The cells of origin from which melanocytes, and consequently their various neoplastic derivatives arise have not been fully defined. However, there are deeply-rooted models suggesting that the postnatal cell source for nevi and melanoma is an epidermal melanocyte. In these models, melanoma is thought to develop through a stepwise loss-of-differentiation process that includes passing through benign nevus stages (1). Further, this model is often illustrated to exclude dermal cells in benign nevi and to pictorially suggest dermal invasion only occurs in invasive melanoma (2-4). Because this model is so well-ingrained, some early small melanomas may be overlooked; primary dermal melanomas may be considered metastatic; and, benign nevi may be unnecessarily excised due to the perception that they are premalignant. The epidermal melanocyte model not only has an impact on patient care, it also impacts basic research. Researchers generally control experiments based on the idea that cell of origin is a differentiated melanocyte and that invasion is a late and uniquely malignant process. Differentiated melanocytes (in the appropriate setting) are best considered as a
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reference for mature cells but using them as cell-of-origin controls may result in data misinterpretation. Thus, it is critical for both patient care and basic research to test and validate models for melanocytic neoplastic development. In this Viewpoint, we will briefly review the spectrum of melanocytic neoplasms, models for cells of origin, and propose experiments that may allow for an improved understanding of the process.

Spectrum of Melanocytic Nevi Human benign melanocytic neoplasms display a broad range of histological and clinical characteristics (5). Congenital melanocytic nevi (CMN) are present at birth comprised of densely packed dermal nevocytes. Nevocytes may also be present in subcutaneous fat, muscles, and central nervous system (5, 6). CMN are most commonly associated with NRAS mutations ; averaging 55% overall (7) but higher for larger lesions (8, 9). However, NRAS mutations are not exclusive to CMN as they can also be seen in acquired melanocytic nevi and are frequently reported in nodular melanoma (7, 10). SOX10 expression also plays a critical role (11). Development of CMN in utero precludes ultraviolet light as the initiating mutagenic factor.

Acquired melanocytic nevi (AMN) begin in childhood and persist into adulthood. All AMN in young children have a dermal component (12). The vast majority of AMN (79%) possess the BRAF V600E mutation (7), which is also the most common mutation in melanoma. Although solar exposure has been shown to increase childhood nevus counts (13), the BRAF mutation does not display a UVB damage signature. Spitz nevi are acquired melanocytic lesions exhibiting spindle and epithelial morphologies with both dermal and epidermal involvement. Spitz nevi are associated with HRAS mutations and amplifications (13%) (7). HRAS mutations are exceedingly rare in melanoma.

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Blue nevi are comprised of heavily pigmented dermal melanocytes with no apparent epidermal connection. Congenital or acquired pigmented dermal melanocytic lesions also exist following neural innervation pathways (Ito/Ota) (5). Blue nevi are most commonly associated with GNAQ/GNA11 (55%/6.5%) mutations (14). Although GNAQ mutations can occur in cutaneous melanoma (15), GNAQ/GNA11 is more commonly associated with uveal melanomas (14). Entirely epidermal melanocytic neoplasias (junctional nevi) and solar induced hyperplasias (solar lentigines) are more common in older individuals (5, 12). Given the diverse clinical characteristics of melanocytic neoplasms, it is possible that they have different cells of origin (cellular differentiation stage and/or tissue location). It is also likely, given molecular characteristics of these tumors, that mutated/active pathways are responsible for the clinical/histologic appearance. A combination of these factors could also be involved with earlier developmental events leading to larger lesions.

Potential cells of origin Given the diverse spectrum of melanocytic neoplasms and complexity of developmental/regenerative melanocytic pathways, several different mechanisms may be involved in nevogenesis. Three models will be reviewed: (1) epidermal melanocytic, (2) dermal precursor, and (3) circulating precursor.

Epidermal melanocytic model Unna first proposed an epidermal origin of nevi termed abtropfung in 1893 (16). Theoretically, nevogenesis commences when an epidermal melanocyte is mutated, proliferates, and produces cells that eventually migrate into the dermis. As nevocytes migrate deeper, melanocytic differentiation markers decrease, size decreases, and they stop proliferating (oncogene induced senescence). A variant of this model has also been applied to melanoma development suggesting malignant transformation involves evolution of a common nevus to a dysplastic nevus and eventually to an intraepidermal (in situ) melanoma prior to developing the malignant capacity to invade the dermis.
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Differentiated and precursor melanocytes exist in adult human epidermis (17, 18). Multipotent epidermal neural crest-derived stem cells (NCSCs) have also been isolated from adult hair follicles (19, 20). A dynamic bidirectional equilibrium has been elucidated in the murine system between melanocytic stem cells and more differentiated transiently amplifying cells (21). Any of these cells could be the cell of origin. Precursor cells provide a source of new epidermal melanocytes as seen in vitiligo recovery and the hair cycle. Additionally, loss of this cell reservoir has been associated with hair graying (22). Developmentally, the epidermis/hair bulge is populated with melanocytic precursor cells that migrate from the dermis (23-27). Reports of gray hair repigmentation suggest that this reservoir can be repopulated under some circumstances (28, 29).

An epidermal origin model is consistent with the pathologic location of junctional nevi, lentigines and in situ melanomas. A particularly interesting case may be made for lentigo maligna melanomas, which are often associated with solar lentigines. It is possible that mutations in keratinocytes (FGFR3/PI3KC) (30, 31) lead to local cytokine alterations promoting melanocytic growth/recruitment in a region with ongoing UV mutagenesis. Although melanocytes likely leave the epidermis via stratum corneum elimination (32-34), dermal migration is possible. While mature adult epidermal melanocytes cannot survive in the dermis (35), dedifferentiation of human melanocytes has been demonstrated in vitro (36) which could allow dermal survival. However, nevus cell skin reconstructs have not yet been able to demonstrate epidermal to dermal migration (37-39). Nevertheless, since epidermal Langerhans cells do migrate into the dermis, lymphatics, and circulatory system, it remains feasible that melanocytes could do the same. However, there are a number of apparent flaws with the epidermal model. All acquired nevi in young children appear to have a dermal component, making it unlikely that growth starts in the epidermis (40). Furthermore, blue nevi lack epidermal involvement and congenital nevi are able to cross divided tissue planes (5), both inconsistent with an epidermal origin. Also, epidermal melanoma models that suggest transition through nevus stages are problematic given that the majority of melanomas appear to develop de
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novo from normal skin (32). Thus, while an epidermal origin is feasible, it is not consistent with clinical findings for many melanocytic neoplasms.

Dermal precursor model Masson was the first to propose a dermal origin of nevi (41). This concept was further refined by Cramer (42) who postulated that melanocyte precursors originating in neural crest mature upwards along peripheral nerves in discrete stages toward the epidermis. Quiescent dermal precursor cells likely accumulate mutations, possibly due to UVA induced oxidative damage or other mechanism. Only specific mutations would be expected to induce nevogenesis ( i.e. BRAF, NRAS, HRAS, and GNAQ) whereby each mutation, when activated, favors a specific growth pattern (common/acquired, congenital, Spitz, and blue nevus, respectively) (7, 32). Rarely, mutations in critical growth controlling genes may also occur along with driving mutations in precursor cells, setting the stage for melanoma development upon activation. The activating signal is unknown but could be inflammatory/cytokine induced (43-50). Once activated, cells would attempt to follow normal developmental/regenerative pathways toward the epidermis; however, the presence of the aforementioned mutation and its interaction with other genetic and environmental factors would cause cellular proliferation/migration in different patterns (32). Mutant cells would migrate to and persist only in favorable locations for their survival. Cellular/tissue interactions could also account for phenotypic variation and heterogeneity. Growth would cease in benign nevi but, due to co-existent growth controlling pathway mutations, would continue in the malignant lesions. Secondary mutations and epigenetic changes could occur in nevi and account for nevus associated melanomas. For malignant tumors, low risk cells would favor the epidermal environment and appear primarily in situ while high risk tumors would grow readily in the dermis. Tumor thickness would reflect the degree of tumor dependence on epidermal cytokines and correlate with risk. After migrating into the epidermis, excess melanocytic cells would be shed into the stratum corneum.

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Schwann cell precursors (SCPs) have recently been shown to give rise melanocytes through the ventral pathway and are likely cell-of-origin candidates for melanocytic neoplasias (51-53). SCP developmental biology and potential relationship to melanocytic disease processes has been recently reviewed (54). In addition, other dermal stem cell populations could certainly play a role (55, 56). Most murine melanoma/nevus models are dermal (57, 58). Developmentally, dermal melanoblasts migrate into the epidermis and increase proliferation (27). This may explain higher proliferative rates in epidermal vs dermal compartments in human lesions. However unlike humans, shortly after birth most epidermal melanocytes are lost in mice. This loss can be prevented by forced expression of kit ligand (kitl)(59), a factor critical in melanocyte homeostasis (60). Kit activated murine melanoblasts do demonstrate dermal to epidermal migration (25). Some murine melanoma models with increased kitl show increased epidermal melanoma involvement (58); however the majority of murine melanoma/nevus models are dermal thus favoring a dermal origin. A dermal origin fits well with most melanocytic neoplasias and is consistent with normal development. While this might seem inconsistent with in situ melanomas, it is noteworthy that on review 29% of in situ lesions contained a dermal component (61), thus it is possible that even these tumors are based on dermal cells migrating into the epidermis.

Circulating precursor model Metastatic cells from epidermotrophic melanomas circulate and reimplant in skin - some even give rise to lesions that appear to be in situ (62). Benign circulating nevocytes have also been noted (63). These examples illustrate the possibility that nevogenesis is a systemic process (64). The source of circulating cells could initially be a cutaneous melanocytic neoplasm. Mutated cells could be washed into the lymph node basin with more stem-like cells passing directly through (65), entering the bloodstream, and migrating back into skin. Alternatively, the source could be a circulating neural/stem cell progenitor (66) - potentially bone marrow derived (67, 68) or a cell from another tissue - mutated in a manner that promotes melanocytic neoplasia.
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A circulating population of nevus progenitor cells implanting at multiple sites could provide an explanation for eruptive nevi and satellite lesions in congenital nevi. It also is consistent with the signature nevus concept (69) due to the same precursor cell population being able to give rise to numerous similar nevi. Given polyclonality (70) noted in nevi, and self-seeding reported for melanoma (71), it is possible that different circulating precursor cells may insert into pre-existing lesions giving rise to heterogenety. The circulating precursor model may seem farfetched but it does offer an explanation for eruptive nevi and for patients with numerous similar nevi. It is also consistent with the clinical presentation of epidermotropic melanoma.

Questions needing answers One unifying model may not hold for all melanocytic neoplasias. Since these models drive both basic research and patient care, it is important that they be tested and validated. Multiple questions remain. What is the differentiation and activation state of the cell of origin? Genetic fate-mapping experiments in mice will probably ultimately answer this question by tagging cell types (i.e. neural crest cells, schwann cells, SCPs, mature melanocytes) with tissue-specific and drug inducible cre/lox and/or flp/frt markers just prior to tumor induction through UV or other mechanisms. The challenge here may be in the plasticity of the system and biases introduced in experimental models such as using melanocytic promoters to drive oncogenic proteins biasing expression to cells with melanocytic differentiation. Another challenge could be labeling quiescent cells given their low levels of transcriptional activity. What is the location and migration direction of the cell of origin? Fate mapping experiments (above) may provide cellular location data. Activation of cells of origin in a specific skin layer might be accomplished through focused two photon or other modalities such as fiber optic probes. Imaging modalities including confocal may help in determining migration direction.

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To what extent can initiated cells remain dormant and what are controlling mechanisms? A quiescent progenitor cell as the target of the initial mutagenic event fits well with the association between childhood sun exposure and melanocytic neoplasms later in life (72, 73). Once the cell of origin has been identified, specific studies could be conducted to address how the number, proliferative potential, and immune tolerance to mutations in the cell of origin change with age. Pathways driving or suppressing activation of the cells could also be studied. These pathways may include inflammatory pathways or cytokines associated with eruptive nevi (50, 74, 75). Pathway suppression may prevent mole and melanoma development.

What is the timing and sequence of mutational events during tumor progression? Many models suggest the initial neoplastic event is a driving mutation such as BRAF V600E , yet BRAF activation causes senescence in melanocytes (76). Given polyclonality (70) noted in nevi, it is possible that driving mutations such as BRAF are secondary to a yet unidentified primary mutation/epigenetic/microRNA (77) change. In melanoma, to prevent senescence, it is likely that cell cycle/survival pathways are mutated before the driving mutations occur. Once cells of origin are identified, it may be possible to initiate them (UV or other means), isolate single cells, expand colonies and assay tumorigenic potential versus underlying induced mutations. The impact of mutation timing could also be studied by utilizing a system in which mutant genes could be turned on in the cell of interest in different orders; for example if BRAF is activated for a prolonged time period before PTEN is inhibited, does this result in a less virulent tumor than if PTEN was inhibited first?

Do melanocytic precursor cells circulate and reintegrate into the skin? As techniques to isolate circulating cells improve, it may become easier to identify rare circulating cells in nevus prone patients. It is possible that patients with specific mole patterns also have specific circulating mutant precursors. Circulating cells with different driving mutations have been noted in

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melanoma patients (78) and self-seeding has noted in metastatic tumors (71); thus it is possible a similar process occurs in patients with polyclonal nevi (70).

Conclusion The cell(s) of origin and subsequent developmental process for melanocytic neoplasias have yet to be fully defined. Data is building to support a dermal origin, possibly an SCP, for most acquired nevi and melanomas but multiple origins remain possible. Ultimately, insight gained from understanding this process will not only help patients with benign and malignant melanocytic processes but it may also impact developmental modeling and care of other solid tumor patients.

Acknowledgments: Anna Fund Melanoma Program, Sylvester Comprehensive Cancer Center, Frankel Family Division of Melanocytic tumors, Department of Dermatology and Cutaneous Surgery, and benefactors especially William Rubin and family/friends.

Author contributions All authors drafted and/or revised the manuscript.

Conflict of interest Grichnik has consulted for Roche, Novartis, CaliberID and DigitalDerm. Other authors note no conflicts.

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Figures Figure 1. Diversity of nevus types. Shown is the clinical presentation and histopathologic features of giant congenital nevus with satellite lesions (A), Spitz nevi (multiple, excision scar also present) (B), common compound nevus (C), blue nevus (D), and a lentigo (E). Dermoscopic images are included as insets for C and D and as the clinical for E. Figure 2. Cell of origin model. Illustrated is a model for the development of melanocytic neoplasias. If the cell of origin is a dermal stem cell (denoted by the *), it can have several potential origins: as a resident of the dermis, as a migrant from the blood stream or as a derivative from a Schwann cell precursor. If this dermal cell of origin is not mutated, it will follow normal developmental pathways when activated (1); however, if mutated, upon activation this dermal stem cell will expand and migrate abnormally in a manner ultimately dependent on its underlying mutation (2). This differential pathway
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of cellular proliferation and growth produces varying growth patterns influenced by the underlying mutation. Additionally, within this model, cells within the dermis could be washed into the lymphatic system to circulate throughout the bloodstream and potentially re-implant elsewhere in the skin (3). Also, neural crest stem cells from the bone marrow could also enter the circulation and relocate to the skin entering into this model as an additional potential precursor source for the dermal cell of origin. Following a more traditional epidermal melanocyte model of nevus development, it is possible that a mutated epidermal melanocyte could migrate into the dermis from a fully differentiated or potentially a more dedifferentiated state (4).

This article is protected by copyright. All rights reserved.

This article is protected by copyright. All rights reserved.