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(2014) 33, 2413–2422 & 2014 Macmillan Publishers Limited All rights reserved 0950-9232/14 www.nature.com/onc

REVIEW Melanoma : new concepts and evolving paradigms

WE Damsky1,2, N Theodosakis1 and M Bosenberg1

Melanoma progression is typically depicted as a linear and stepwise process in which metastasis occurs relatively late in progression. Significant evidence suggests that in a subset of melanomas, progression is much more complex and less linear in nature. Epidemiologic and experimental observations in melanoma metastasis are reviewed here and are incorporated into a comprehensive model for melanoma metastasis, which takes into account the varied natural history of melanoma formation and progression.

Oncogene (2014) 33, 2413–2422; doi:10.1038/onc.2013.194; published online 3 June 2013 Keywords: ; melanoma; metastasis; BRAF; b-catenin

INTRODUCTION observations from human melanoma patients which add temporal Melanoma rates are rising worldwide.1 Most morbidity and and spatial complexity to metastasis. Many melanoma patients are mortality in melanoma is associated with metastatic disease, cured after excision of their primary tumor; however, some are which can occur even from thin primary tumors.2 Accordingly, not, and will have disease recurrence. Melanoma recurs less metastasis is a particularly ominous sign; when metastasis is frequently at the site of the original primary tumor, instead more clinically evident, is very poor. For example, melanoma commonly recurring at different sites in the body as metastatic patients with three or more sites of metastatic disease die within lesions.9 These recurrence patterns suggest that melanoma cells 1 year 495% of the time.3,4 New targeted and immunomo- had already spread before excision of the primary tumor, even dulatory therapies such as and have though this spread might not have been clinically apparent at offered the first improvements in overall survival to melanoma that time. In melanoma patients, metastatic recurrence can patients in several decades.5,6 Unfortunately, only a subset of occur within a relatively short period of time, but takes 45 patients benefit in a durable fashion from these therapies, while years in about 40% of patients.4,10–12 The period in between initial most will still ultimately succumb to metastatic disease. excision of the primary tumor and subsequent metastatic Melanoma results from the malignant proliferation of melano- recurrence is often referred to as metastatic dormancy, where cytes, the normal pigment producing cells in skin. Classically previously disseminated tumor cells are thought to persist in a melanoma progression and metastasis has been depicted using relatively non-proliferative state. the Clark model.7 In this model, progression of normal The mechanisms regulating metastatic dormancy are not to metastatic melanoma is linear, and consists of a particularly well understood, however, several hypotheses have series of steps including: benign precursor lesion formation been proposed. One hypothesis suggests that tumor cell extrinsic (melanocytic ), , progression through both factors such as the actively and purposefully radial and vertical growth phases of primary tumor growth, and constrain growth of disseminated cells.13,14 Another model ultimately metastasis (Figure 1). The accumulation of genetic and suggests that a relative inability of disseminated tumor cells to epigenetic changes is thought to drive progression through these recruit new blood vessels limits frank outgrowth of disseminated steps.8 In the Clark model, metastasis ensues late in disease disease.15 Finally, some authors have proposed that metastatic progression, only after all previous steps have been completed tumor cells simply growth arrest in these new stromal and relatively rare clones with metastatic competency emerge. microenvironments due to a relative mismatch between cell–cell Although this model likely depicts the natural history of some and cell–extracellular matrix interactions.16,17 Despite differences melanomas, several pathologic, epidemiologic, and experimental in the primary mechanism enforcing dormancy, these models are observations suggest that the progression of other melanomas similar in that they hypothesize that disseminated tumor cells must may be more complex and less linear in nature. Evolving overcome some barrier(s) before progression to a macrometastatic conceptual paradigms of melanoma progression that account lesion. Metastatic dormancy and subsequent reactivation of for these additional observations and incorporate the many growth in melanoma have been recently reviewed.12,16,18 individual steps in metastasis will be explored here. The seed-and-soil hypothesis of metastasis underscores the additional spatial complexity of metastasis. Seed-and-soil posits that patterns of metastasis observed in human TEMPORAL AND SPATIAL COMPLEXITY OF METASTASIS result from inherent compatibilities (or incompatibilities) between Metastasis involves a series of steps which must be completed different tumor cell types and different sites of metastasis.19 before the emergence of clinically meaningful metastatic disease For example, has an extraordinary proclivity for (Figure 1). These steps are further complicated by additional metastasis to the liver. Nearly 90% of patients with metastatic

1Department of , Yale University School of Medicine, New Haven, CT, USA and 2Department of , University of Vermont College of Medicine, Burlington, VT, USA. Correspondence: Dr WE Damsky, Department of Pathology, University of Vermont College of Medicine, Given Box A14, 189 Beaumont Avenue, Burlington, VT 05495, USA or Dr M Bosenberg, Department of Dermatology, Yale University School of Medicine, 333 Cedar Street, LCI 501, PO Box 208059, New Haven, CT 06520, USA. E-mail: [email protected] or [email protected] Received 2 March 2013; revised 22 April 2013; accepted 22 April 2013; published online 3 June 2013 Melanoma metastasis WE Damsky et al 2414

Figure 1. Melanoma formation and progression. Melanoma formation and metastasis are complex processes. Primary tumor formation has classically been depicted using a linear model in which normal melanocytes progress through several precursor lesions and ultimately to melanoma (individual steps colored in blue). However, substantial evidence suggests that such linear progression is only one pathway to and that not all steps always occur during progression of individual melanomas. Other pathways to primary tumor formation are also depicted (non-linear). There is additional evidence to suggest that dissemination of cells away from the primary lesion may not be a unique feature of invasive melanomas. The possibility of spread from these lesions is depicted with question marks. Approximately 4–12% of metastatic lesions are not associated with a primary cutaneous melanoma (depicted in orange). The individual steps in metastasis are depicted in green. Unlike primary tumor formation, steps in metastasis are likely to always occur in a linear fashion; although it is unclear whether passage of tumor cells through lymphatics is absolutely required for systemic dissemination. Direct entry of tumor cells into venous return may also be possible and is depicted with a question mark. The from micrometastasis to macrometastasis is likely a critical, rate-limiting step in the of many disseminated melanocytic lesions. This transition may take months to years (if it occurs at all) and is preceded by metastatic dormancy. Macroscopic metastatic lesions are thought to be able to seed additional metastases (21 spread). It is unclear whether 21 spread can also occur from micrometastatic lesions, which is depicted with a question mark.

uveal melanoma will have metastatic disease in the liver, epithelial cancers have even demonstrated that spread of which is often the only site of metastasis.20 In cutaneous abnormal cells can precede primary tumor formation.23,24 These melanomas, this principle is also important with common sites disseminated epithelial cells are capable of persisting and of clinically detectable visceral metastasis including: lung, brain, subsequently transforming to overt metastatic growths at these liver and bone, while metastases to other sites are either less distant sites.23,25 In this context, it is notable that 4–12% of common or rarely encountered clinically.3 Even among common patients with metastatic melanoma do not have a clinically sites of melanoma metastasis, certain epidemiologic patterns identifiable primary tumor.26–28 There are different explanations suggest additional biological complexity. For example, liver for this observation, though one is that some of these metastatic metastasis and are inversely correlated.3 Our lesions represent malignant progression of abnormal melanocytes current understanding of the molecular mechanisms underlying that had spread previously. melanoma metastasis to different organs has been recently In fact, non-malignant melanocytes are routinely visualized reviewed.21 outside of their normal epidermal location in humans. In compound melanocytic nevi, benign proliferations of melanocytes are found within the , demonstrating that spread through PREMALIGNANT DISSEMINATION AND PARALLEL the is not a unique feature of malignant PROGRESSION cells. Melanocytes can even be visualized in a subset of lymph In traditional models of metastasis, spread to spatially restricted nodes from normal, non-melanoma bearing patients.29–32 These sites is thought to be a unique feature of a subset of particularly melanocyte clusters in normal lymph nodes are referred to as aggressive tumor cells that arise late in the course of disease. nodal nevi. Interestingly, many nodal nevi bear BRAFV600E- However, some observations in melanoma and other cancers activating .33 BRAFV600E mutations are also found in argue that spread of tumor cells can occur much earlier during most benign cutaneous melanocytic nevi (mutations in melanoma primary tumor formation. For example, kinetic models of uveal are discussed in more detail below),34 leading some authors to melanoma based on tumor doubling times estimate that on speculate that nodal nevi could represent cells that have spread average, metastatic spread of tumor cells to the liver is initiated up from these benign lesions.35 However, it is very difficult to to 5 years before identification and removal of the primary definitely exclude the possibility that nodal melanocytes are tumor.22 Genetically engineered mouse models (GEMMs) of instead remnants of embryonic development, or embryonic rests.

Oncogene (2014) 2413 – 2422 & 2014 Macmillan Publishers Limited Melanoma metastasis WE Damsky et al 2415 Accordingly, direct evidence supporting early dissemination of Although the prevalence of MITF and b-catenin dysregulation in metastatic cells in melanoma is currently absent. melanoma is clear, the effect of this dysregulation with respect to The hypothesis that disseminated precancerous or even frankly melanoma progression and metastasis is debated. Increased MITF malignant cells can persist at distant sites and retain the potential levels and/or levels of melanocytic differentiation antigens in to progress to clinically relevant metastatic disease is often referred human melanomas have been associated with both a relatively to as the parallel progression model of metastasis.36,37 The parallel good outcome58,59 and a relatively poor outcome.60–62 Similarly, progression model has significant conceptual overlap with attempts to correlate b-catenin levels with outcome have been metastatic dormancy. In this model, continued genetic and/or challenging to interpret. Most consistently, increased b-catenin epigenetic evolution of tumor cells at distant sites facilitates has been associated with an improved outcome in some larger progression to overt metastasis. A prediction of the parallel studies, particularly when combined with additional markers into progression model is that DNA sequence and/or epigenetic prognostic models.63–65 Some smaller, previously published modifications should be relatively disparate between primary and reports did not find this relationship between increased metastatic lesions if continued evolution at distant sites is required b-catenin levels and poor outcome and some suggested, rather, for progression to overt metastasis. Recent efforts comparing DNA that b-catenin dysregulation is associated with disease sequence between matched primary and metastatic tumors in a progression and metastasis.52,66,67 Of course, correlating patient handful of melanoma patients have shown both a relative outcome with levels or staining patterns is difficult to homogeneity38,39 and a relative heterogeneity40 between interpret at a mechanistic level. b-catenin is thought to have matched lesions. Similar studies in epithelial cancers have more different functions depending on its subcellular localization, so commonly reported relative homogeneity between matched levels in tissue likely do not accurately represent functional primary and metastatic lesions, but significant heterogeneity in activity. Finally, prognosis is complex and represents several DNA sequence has also been reported.41 These observations aspects of disease biology, not only the likelihood of metastasis. suggest that relatively early dissemination with parallel progression Unfortunately, studies designed to functionally evaluate the and relatively late dissemination with limited evolution at distant roles of MITF and b-catenin in melanoma progression have also sites are both pathways with the potential to give rise to clinically been difficult to interpret as a whole.68 For example, increased meaningful metastatic disease. However, as it is not clear if- and/or levels of MITF have been shown to both increase69,70 and which- of these additional genetic changes have a functionally decrease71–74 metastasis and/or surrogate measures of important role in driving disease progression, these conclusions metastasis in experimental models of melanoma. Similarly, based on sequencing data are still fairly speculative. The degree to seemingly contradictory results are found with respect to the which early dissemination and parallel progression contribute to consequences of increased b-catenin in melanoma progression, metastatic melanoma pathogenesis on a population scale remains with some studies describing a pro-progression role for b-catenin to be seen. and others describing a progression restricting role.75–78 In an attempt to reconcile these observations, several hypo- theses have been proposed. Some groups have suggested that DIFFERENTIATION IN METASTASIS a relative balance in the degree of melanocytic differentiation is The degree and type of differentiation of tumor cells with respect important in driving metastasis, where both too much or too to their ability to metastasize is perhaps the most well-studied little can restrict progression.75,79 A separate hypothesis, the relationship in metastasis research. Epithelial–mesenchymal phenotypic plasticity (or phenotype switching) model, proposes transition is a model of metastasis developed in the study of that transient changes in the degree of melanocytic differentiation epithelial cancers, which posits that relative loss of epithelial in a particular melanoma may help to facilitate the different steps differentiation and relative gain of mesenchymal characteristics of metastasis for which they are advantageous.80–82 In this model, facilitates early steps in metastasis, such as local tissue increased melanocytic differentiation is thought to drive local and intravasation.42 Despite being a widely accepted paradigm for proliferation, while relative loss of melanocytic differentiation is cancer metastasis, epithelial–mesenchymal transition is not thought to facilitate the metastatic spread itself. This model is without controversy, even within epithelial cancers.43 Although somewhat consistent with recent studies in epithelial cancers that melanocytes share some features of differentiated epithelial cells, suggest different cellular programs drive the distinct processes of they are derived from the neural crest and have a differentiation tumor cell spread and tumor cell proliferation at distant sites.83,84 program related to pigment production,44 suggesting that the Other groups, including ours, have suggested instead that relationship between differentiation and metastasis in melanoma increased melanocytic differentiation has context-specific effects may be even more complex. with respect to progression and metastasis. A recent study by In melanocytes, differentiation is largely controlled by micro- Gallagher et al.85 suggested that while b-catenin activation in phthalmia-associated (MITF) and WNT/ melanocytes restricts cellular motility and migration, b-catenin b-catenin signaling. MITF is a transcription factor central to activation in melanomas enhances metastasis when measured melanocyte survival, proliferation and -pigment in a GEMM. Data from additional melanoma GEMMs production.45 Canonical WNT signaling through b-catenin also developed in our lab also suggests that the effects of b-catenin promotes increased melanocytic differentiation through direct and Mitf may be context-dependent.86 In these GEMMs, the effect transcriptional upregulation of MITF.46 b-catenin is also required of increased melanocytic differentiation on metastasis was for melanocyte survival47 and differentiated melanocyte dependent on concurrent genetic changes within the tumor. For function.48 Both MITF and b-catenin signaling are dysregulated example, in GEMMs with mutant Braf and inactivated Pten, in human melanomas. MITF is amplified in 10% of primary b-catenin substantially increased metastasis to the lungs, while in melanomas and 21% of metastases.49 Germline MITF mutations other GEMMs with wild-type Pten, b-catenin seemed to promote that alter MITF function are associated with an increased local tumor growth, but not metastasis.86 lifetime risk of melanoma.50,51 Oncogenic b-catenin mutations A last example of the additional layers of complexity in are found in B5–7% of melanomas and are thought to lead discerning the effect of b-catenin in melanoma progression is to constitutive activation of the canonical WNT pathway.52–55 illustrated by the recent observation that b-catenin in melanoma More broadly, WNT/b-catenin pathway activation can occur cells has substantial immunosuppressive effects in the local tumor through a variety of mechanisms in addition to of microenvironment.87 Assays that study melanoma metastasis, in b-catenin,56 with evidence of pathway activation in at least 1/3 of for example, an immunodeficient mouse model may not account melanomas.57 for these potential tumor-extrinsic effects of b-catenin activation.

& 2014 Macmillan Publishers Limited Oncogene (2014) 2413 – 2422 Melanoma metastasis WE Damsky et al 2416 Context is of critical importance when studying metastasis in which have reciprocal interactions with tumor cells. Tumor mouse models whether it is genetic background of the tumor, microenvironments vary during different steps in progression immune status of the host or another variable. A variety of and metastasis and can both inhibit and facilitate progres- approaches and mouse model systems have been employed, sion.16,17,41,88 In melanoma, changes in the tumor micro- many of which are summarized in Figure 2. Importantly, these environment can have very powerful effects on tumor cells. approaches do not study metastasis in a broadly comparable way, For example, embryonic and skeletal muscle microenvironments an additional consideration that must be taken into account when can restrict both proliferative and metastatic phenotypes.89,90 interpreting experimental data regarding metastasis. Context is In mouse models of melanoma metastasis, the microenvironment also of critical importance when interpreting staining patterns in at distant sites is thought to be critical in eliminating disseminated human melanomas. Staining patterns for a particular protein in tumor cells and preventing both formation of micrometastases, as primary versus metastatic, treated versus untreated or PTEN wild- well as progression of micrometastases to macrometastases.91 type versus PTEN mutant melanomas, for example, may not be Interestingly, the microenvironment can also positively influence broadly comparable. MITF and b-catenin in melanoma serve as an tumor progression. In epithelial cancers, for example, loss of the important example of these context-specific considerations in the transforming growth factor-bII receptor in stromal fibroblasts can study of metastasis. Mouse models and the particular advantages initiate neoplasia in otherwise unmodified epithelial cells.92 of GEMMs will be discussed further below. Immune cells are one particularly well-studied component of the in both melanoma and other cancers. Although the effects of the immune system in melanoma TUMOR MICROENVIRONMENT AND MACROENVIRONMENT will not be reviewed in detail here, in general, adaptive immunity Tumor microenvironments are defined by both cellular and is thought to help constrain progression of both primary and non-cellular constituents in the tissue immediately surrounding metastatic disease.14,93 Components of the innate immune system tumor cells. These factors include: stromal and immune cells, such as macrophages, on the other hand, have been shown to extracellular matrix, soluble factors and mechanical forces, all of facilitate both primary tumor progression and metastasis in a

Figure 2. Approaches for studying metastasis in mice. A variety of methods are used to study metastasis in mice, the most common of which are depicted here. Each of these approaches has relative advantages and disadvantages, including which individual steps of metastasis can be studied using the approach. The steps that can be studied using each method are shown as colored boxes. The immune status of the mouse host is also an important consideration. Immunodeficient mouse models do not account for the effect of the immune system on progression, while immunocompetent mouse models do not allow for the use of human melanoma cells. (a) In orthotopic models, melanoma cells are injected directly into the desired site of metastasis (i.e., the brain) leading to formation of a mass. Either human or murine melanoma cells can be injected into an immunodeficient mouse. Only murine melanoma cells can be injected into an immunocompetent mouse, which must be syngeneic (i.e., B16 melanoma cells into a c57bl/6 mouse). (b) Melanoma cells can be injected intravenously to simulate metastasis. With this approach either human or murine melanoma cells can be injected into an immunodeficient mouse. Murine melanoma cells can be injected into an immunocompetent, syngeneic mouse. (c) Melanoma cells can be injected subcutaneously leading to formation of a mass, which can subsequently metastasize to other sites. This approach allows for the use of either human or murine melanoma cells in immunodeficient mice or murine melanoma cells in immunocompetent, syngeneic mice. (d) GEMMs are based on the introduction of genetic changes commonly found in human melanomas, such as BrafV600E, specifically to the melanocytes of mice. Appropriate combinations of genetic changes result in the spontaneous formation of melanomas from endogenous melanocytes in an immunocompetent mouse. GEMMs recapitulate all steps of melanoma pathogenesis; however, study of human melanoma cells is not possible with GEMMs.

Oncogene (2014) 2413 – 2422 & 2014 Macmillan Publishers Limited Melanoma metastasis WE Damsky et al 2417 variety of experimental assays.94 In human melanomas, increased mutations to BRAF and NRAS are also found in the majority of macrophage staining is associated with poor outcome.95 The benign melanocytic nevi, suggesting that these changes represent relationship between the immune system and melanoma relatively early, although not independently sufficient, events in metastasis has been recently reviewed.96 melanomagenesis. Even the earliest genetic changes in primary Tumor cell extrinsic factors that act in an organism-wide tumor formation have been postulated to have a functionally fashion are also likely to be important in melanoma metastasis. important role in metastasis,114 which is consistent with the Perhaps the most well-known example of this type of ‘macro- observed early dissemination of abnormal cells in epithelial cancer environmental’ effect is the premetastatic niche. In this model, GEMMs discussed above. The hypothesis that early genetic soluble tumor-derived factors circulate systemically and can enact alterations drive many aspects of tumor progression is also complex programs that alter distant tissues in a way that facilitates consistent with the observation in human melanomas that later metastasis to those sites.97 Recent work has implicated proliferation rate115,116 and thickness4 of primary tumors are tumor-derived exosomes containing melanocytic antigens in very strongly correlated with the likelihood of metastasis. initiating premetastatic niche formation in melanoma.98,99 Epidemiologic observations from human melanomas suggest Other examples of interactions between established, spatially that there is no difference in rates of metastasis between BRAF and separated malignant lesions through endocrine-like mechanisms NRAS mutant melanomas,117,118 though both may exhibit higher have also been described,100,101 though the relative importance rates of metastasis than RAS/RAF wild-type melanomas.119 of these observations in human melanoma metastasis remains Interestingly, a recent study suggested that BRAFV600K-driven unclear. Additional macroenvironmental factors such as systemic melanomas may be more metastatic than BRAFV600E-driven inflammation,102–104 organismal metabolism,105–108 host genetic melanomas,120 suggesting that the metastasis promoting effect background109–111 and even male gender112 can all influence the of NRAS and/or BRAF may be more complex than mutation time course of progression and pattern of metastasis. An presence or absence alone. improved understanding of these macroenvironmental changes Functional studies of mutant RAS and RAF have reinforced the and the specific manner in which they affect tumor and tumor role of these in metastasis. For example, melanoma microenvironmental biology will be important moving forward. GEMMs have shown that NRAS mutant melanomas are inherently more metastatic than melanomas driven by other mutant RAS isoforms.121,122 In other mouse models, inhibition of mitogen- MUTATIONS AND METASTASIS activated protein /extracellular-signal-regulated kinase Our understanding of the genetic alterations commonly found in signaling can block lung metastasis.118 Although a specific human melanomas has advanced significantly over the past mechanism by which RAS or RAF mutation drives metastasis in several years (Figure 3). The mitogen-activated protein kinase/ melanoma is unclear, recent studies have proposed mechanisms extracellular-signal-regulated kinase pathway is the most com- that relate BRAF mutation to cell invasiveness.123,124 monly mutated pathway in melanoma, with BRAF and NRAS being The PI3K/AKT pathway is also frequently constitutively activated the most commonly mutated components.113 Activating in melanoma, most commonly through genetic or epigenetic

Figure 3. Recurrent alterations to key signaling pathways in melanoma. Proteins depicted in green are recurrently activated in melanoma. Mechanisms for activation include specific gain-of-function mutations, DNA copy number gains, and/or overexpression. Proteins depicted in blue are recurrently inactivated in melanoma. Mechanisms of inactivation include: point mutations, genomic losses and epigenetic silencing. Proteins depicted in gray are key effectors of pathways that are frequently hyper-activated in melanoma, but are not themselves commonly intrinsically dysregulated. Various negative feedback loops exist within and between pathways, though these are not depicted for simplicity. *Germline MC1R polymorphisms are related to altered pigment production and increased risk of melanoma. **Mutations occur in the TERT promoter rather than in coding DNA sequence.

& 2014 Macmillan Publishers Limited Oncogene (2014) 2413 – 2422 Melanoma metastasis WE Damsky et al 2418 inactivation of the PTEN tumor suppressor125,126 and copy number cells never progress to a macroscopic metastasis.153 Together, gains or overexpression of AKT3.127,128 PTEN has been shown to these experimental observations begin to shift the focus towards suppress melanoma metastasis in experimental assays.129 later steps in metastasis. Melanoma GEMMs support a critical role of Pten loss and Patterns of metastasis observed during autopsies of melanoma subsequent activation of the PI3K/Akt pathway in driving patients suggest that not all disseminated melanoma leads to metastasis of melanomas initiated by activating Ras and Raf clinically relevant metastatic disease. Metastatic tumor distribution mutations.130,131 In these models, however, activation of PI3K/Akt is often much more substantial than can be appreciated clinically is also a critical event in primary tumor formation, making specific while patients are alive. For example, intestinal metastasis is effects on metastasis difficult to infer. In additional melanoma detected in only 1–7% of patients clinically, but 26–58% of GEMMs, b-catenin-stabilizing mutations and Lkb1-inactivating patients will show evidence of intestinal metastasis at autopsy.3 mutations have been linked with increased metastasis and are Similarly, liver metastasis, while clinically evident in only 10–20% discussed further below. of patients with metastatic melanoma, is found in 54–77% In the past few years, genome-wide sequencing efforts patients at autopsy.3,154,155 In fact, it appears that subclinical have identified several new recurrent mutations in melanoma. melanoma metastases can manifest in almost any part of the Some of these mutations include: RAC1,55,132 PREX2,55 GRM3,133 body, including sites rarely encountered clinically.3,155,156 More GRIN2A133 and the TERT promoter.134 Discerning the functional sensitive assays for detecting disseminated tumor cells also consequences of these and other recurrent genetic aberrations in suggest that clinically inevident tumor burden may be much melanoma have the potential to uncover additional pathways higher than can be appreciated in melanoma patients. For regulating progression and metastasis (Figure 3). For example, example, micrometastatic disease can be detected by reverse PREX family guanine-nucleotide exchange factors have recently transcription–PCR and immunohistochemistry in large proportion been shown to be required for melanoma metastasis in a of patients and often in lieu of clinically detectable metastatic mouse model.135 Additional efforts have led to identification of disease.157 recurrent BAP1 mutations in the majority of metastatic uveal Other potential clues that growth at distant sites, rather than melanomas.109,136 simply arriving at distant sites, may be a critical rate-limiting step Other large scale approaches have been undertaken in an in the pathogenesis of melanoma metastasis comes from analysis attempt to identify metastasis regulators in melanoma by of draining lymph nodes from melanoma patients. The presence comparing DNA copy number changes137–139 and global mRNA or absence of tumor cells in sentinel lymph nodes has important expression between primary and metastatic melanomas.140 prognostic implications for melanoma patients.4 However, it has Unfortunately, to date these analyses have had little overlap in been shown that melanoma patients with histologic evidence of identified.140 Several other molecular mediators of melanoma spread to lymph nodes, but with tumor cell foci melanoma metastasis have been identified by a variety of o0.1 mm in size, have an equivalent prognosis to patients without approaches and include: Kiss-1,84 NEDD9,141 RhoC,142 MDA-9/ any evidence of spread to lymph nodes at all.158–160 Interestingly, syntenin,143 NM23144 and BRMS1,145 among others.146 complete removal of the draining bed does not improve survival in melanoma patients with evidence of spread.161,162 These observations may suggest that the presence RATE-LIMITING STEP IN MELANOMA METASTASIS? of tumor cells in the draining lymph nodes reflects two aspects of Metastasis is a complicated process with many different steps tumor biology: (1) the ability to disseminate and persist in distant (Figure 1). Traditionally, metastasis research has focused on early sites, and (2) the ability to progress to more clinically meaningful steps in metastasis such as: acquisition of cellular motility, local lesions at these sites. These additional observations, though tissue invasion and intravasation. Conversely, relatively less certainly somewhat speculative, may help to begin to synthesize emphasis has been placed on later steps in metastasis such as: newer experimental data with patterns of metastasis in human extravasation, survival, and both persistence and proliferation at melanoma patients. distant sites. Although these later steps in metastasis are These many considerations emphasize the importance of later inherently more difficult to study, observations in human steps in metastasis and suggest that progression of clinically melanoma patients suggest that understanding these processes undetectable disseminated melanoma to clinically detectable is critical for a comprehensive understanding of the pathogenesis macrometastatic disease may be a particularly important target for of metastatic melanoma. continued development of new adjuvant therapeutic approaches. Early metastasis work in the 1970s, suggested that dissemina- For example, bisphosphonate therapy, a strategy to make bone tion of malignant cells from a primary tumor is a fairly common microenvironments less hospitable to disseminated tumor cells, is event, with 41 million cells estimated to be shed per gram of now being used in an attempt to eliminate latent micrometastatic tumor per day.147 In addition to being relatively common, it was disease in the bones of some breast and prostate cancer also found that circulating tumor cells do not necessarily predict patients.163,164 Analogous, non-conventional adjuvant strategies whether metastasis has already or will ever occur.148 Despite more based on improved understanding of disseminated tumor cell recent technological improvements in the detection of circulating biology may be useful in targeting dormant micrometastatic tumor cells, newer studies still have not consistently found that disease in human melanoma patients. the presence of circulating tumor cells is related to outcome in melanoma.149 Further complicating this picture, and consistent with the early dissemination hypothesis, benign melanocytes and MOUSE MODELS nevus cells are routinely detected in the circulation of patients Mouse models have been critical in advancing our understanding without melanoma.150,151 of metastasis. Mouse models offer the ability to study different Studies tracking the fate of circulating tumor cells in experi- steps of cancer metastasis in a relatively physiologic manner.165 mental models of melanoma metastasis suggest that the Mice have been employed in a variety of different ways to study persistence of disseminated tumor cells at distant sites, the metastasis, each with intrinsic advantages and disadvantages progression of individual tumor cells to micrometastatic clusters of (Figure 2). In general, GEMMs are thought to particularly cells and the outgrowth of micrometastasis to clinically relevant accurately recapitulate the complex biology of tumor initiation, macrometastases are relatively rate-limiting events in metastasis progression and metastasis compared with other in vivo pathogenesis.100,152 Other studies using a variety of different approaches.166 In melanoma GEMMs, the introduction of approaches have also suggested that most disseminated tumor genetic changes specifically to endogenous melanocytes of mice

Oncogene (2014) 2413 – 2422 & 2014 Macmillan Publishers Limited Melanoma metastasis WE Damsky et al 2419 results in spontaneous progression of melanocytes to metastatic 6 Hodi FS, O’Day SJ, McDermott DF, Weber RW, Sosman JA, Haanen JB et al. melanoma in a background that is otherwise unbiased and Improved survival with ipilimumab in patients with metastatic melanoma. N Engl 166–168 unmanipulated. Further, GEMMs are not immuno- JMed2010; 363: 711–723. compromised and incorporate potentially critical effects of the 7 Clark Jr WH, Elder DE, Guerry Dt, Epstein MN, Greene MH, Van Horn M. A study of immune system in disease progression. Recently developed tumor progression: the precursor lesions of superficial spreading and GEMMs offer high rates of metastasis and will likely melanoma. Hum Pathol 1984; 15: 1147–1165. serve as new tools for in-depth study of the mechanisms of 8 Miller AJ, Mihm Jr MC. Melanoma. N Engl J Med. 2006; 355: 51–65. metastasis.86,169 In the Pten/Braf/b-catenin model developed in our 9 Balch CM, Urist MM, Karakousis CP, Smith TJ, Temple WJ, Drzewiecki K et al. Efficacy of 2-cm surgical margins for intermediate-thickness melanomas (1 to lab, very high rates of spontaneous metastasis occur reproducibly 86 4 mm). Results of a multi-institutional randomized surgical trial. Ann Surg 1993; to lymph nodes and lungs. 218: 262–267 (discussion 7–9). New GEMMs such as the Pten/Braf/b-catenin model offer unique 10 Crowley NJ, Seigler HF. Late recurrence of malignant melanoma. Analysis of 168 opportunities for the study of metastasis. For example, incorpor- patients. Ann Surg 1990; 212: 173–177. ating fluorescent reporters into these models will allow for an 11 Slingluff Jr CL, Dodge RK, Stanley WE, Seigler HF. The annual risk of melanoma unprecedented ability to visualize and purify cells from various progression. Implications for the concept of cure. Cancer 1992; 70: 1917–1927. steps of experimentally unmanipulated spontaneous metastasis. 12 Pierard GE, Pierard-Franchimont C, Reginster MA, Quatresooz P. Smouldering Intravital imaging technologies allow real-time visualization of malignant melanoma and metastatic dormancy: an update and review. Dermatol these processes and have been recently reviewed.170 Processes Res Pract 2012; 2012: 461278. 13 Koebel CM, Vermi W, Swann JB, Zerafa N, Rodig SJ, Old LJ et al. Adaptive such as early dissemination of abnormal cells, metastatic immunity maintains occult cancer in an equilibrium state. Nature 2007; 450: dormancy and progression of micrometastatic lesions to frank 903–907. metastases can be studied and manipulated in these models. 14 Eyles J, Puaux AL, Wang X, Toh B, Prakash C, Hong M et al. Tumor cells disseminate early, but immunosurveillance limits metastatic outgrowth, in a mouse model of melanoma. J Clin Invest 2010; 120: 2030–2039. CONCLUSIONS AND FUTURE DIRECTIONS 15 Folkman J. 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