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Molecular biology in conjunctival melanoma and the relationship to mucosal melanoma

2020; Wiley; Volume: 98; Issue: S115 Linguagem: Inglês

10.1111/aos.14536

1755-3768

Lauge Hjorth Mikkelsen,

Immunotherapy and Immune Responses

Conjunctival melanoma is a rare malignancy of melanocytes located in the conjunctiva – the mucosal membrane covering the eye. The course of disease is often uncomplicated with proper treatment; however, a subset of patients develop metastases that can be difficult to treat and are associated with a poor prognosis. Unlike all other mucosal melanoma, conjunctival melanomas are partly exposed to sunlight, similar to cutaneous melanoma. Previous studies of mucosal melanomas have not included conjunctival melanomas, and studies of conjunctival melanomas have mostly been compared with cutaneous melanoma. Therefore, the relationship between conjunctival melanoma and other mucosal melanoma remains fairly unknown. In the era of precision medicine, prognostic markers and therapeutic targets remain of high importance in cancer management. However, in conjunctival melanoma, no markers for metastatic disease exist at the moment. In order to contextualize conjunctival melanoma as a mucosal melanoma, we conducted a series of studies, including a literature review investigating the clinical and molecular characteristics of conjunctival melanoma (I) and an original study investigating the genetic and molecular profile in conjunctival and other mucosal melanomas (II). Furthermore, we investigated whether miRNA could be used as a prognostic marker for metastasis in conjunctival melanoma (III). We identified conjunctival melanoma as being the most frequent mucosal melanoma with a fairly good prognosis overall. We found that conjunctival melanoma has a distinct molecular profile when compared to other mucosal melanomas. This difference was highlighted by frequent mutations in BRAF and a differential expression of 41 genes. Interestingly, sun exposure could only in part explain this difference at the gene expression level. When investigating miRNAs as potential prognostic markers, we observed that primary conjunctival melanomas with and without metastatic potential separated at the miRNA level; however, we were not able to identify one specific miRNA that was predictive for metastatic disease. In conclusion, conjunctival melanoma is a special variant of mucosal melanoma that shares only some characteristics with cutaneous melanoma. miRNA could not be confirmed to be a marker of metastases, and future studies of multiple markers are needed in order to predict metastatic behaviour. Conjunctival melanoma is a rare cancer that mainly affects elderly Caucasians (Larsen et al. 2015). Melanoma is a malignant transformation of melanocytes, and consequently, melanoma may be found at all anatomical locations where melanocytes are present (Mikkelsen et al. 2016). These locations include skin melanoma (91%), ocular melanoma (uveal tract and conjunctiva) (5%), other mucosal melanoma (1%), and melanomas with unknown site of primary tumour (3%) (Chang et al. 1998). Of the ocular melanomas, 85% are constituted by melanomas of the uveal tract and 15% are conjunctival melanomas (Osterlind 1987; Chang et al. 1998; Seregard 1998; McLaughlin et al. 2005). Being in fact a mucosal melanoma, a special feature of conjunctival melanoma is their exposure to sunlight. Most conjunctival melanomas are located in the sun-exposed bulbar conjunctiva, and conjunctival melanoma located in the fornical and tarsal conjunctiva is generally not considered to be sun exposed (Mikkelsen & Heegaard 2018). In contrast, mucosal melanomas of other sites are completely sun-shielded. Interestingly, extrabulbar conjunctival melanoma has been shown to be associated with a poor prognosis more similar to that of mucosal melanomas from non-sun-exposed locations (Larsen et al. 2015; Mikkelsen et al. 2016). Whether this is due to a delayed diagnosis or a molecular similarity to other mucosal melanomas that generally carry a grave prognosis is unknown; however, studies of mutations in the BRAF gene suggest the latter being the case (Larsen et al., 2016a,b; Mikkelsen & Heegaard 2018). In recent years, the molecular biology of cutaneous melanocytic lesions and in particular cutaneous melanoma have been studied in great detail. In particular, a great deal of work has been done in exploring the genetic landscape of cutaneous melanoma, which is clearly reflected in the recent World Health Organization (WHO) classification of skin tumours fourth edition (2018). In this volume, malignant melanoma is separated into different subgroups according to their pathway from melanocyte to malignant melanoma, based on genetic profile and precursor lesion. Regarding conjunctival melanoma, only few molecular studies have been carried out. Mutations in the BRAF, NRAS, and NF1 genes have been identified (Spendlove et al. 2004; Beadling et al. 2008; Wallander et al., 2011; Alessandrini et al. 2013; Griewank et al. 2013a,b; Sheng et al. 2015; Larsen et al., 2016a,b; Scholz et al. 2018), but still, none of these seem to correlate with the course of disease or prognosis (Larsen 2016). In particular, the metastatic pattern is puzzling – with all from multiple local recurrences to very late occurring distant metastases being seen (Larsen 2016). In this series of studies, we investigated conjunctival melanoma at different molecular levels, including genetics, gene expression, and epigenetics, in order to provide a piece of the conjunctiva melanoma puzzle. The conjunctiva is innervated by the ophthalmic division of the trigeminal nerve (V1), mainly through branches from the nasociliary nerves, lacrimal nerve, frontal nerve, and the infraorbital nerve. Close to the limbus, the mucous membrane is innervated by the ciliary nerves (Duke-Elder & Wybar 1961). The arterial supply is provided from the palpebral branches of the nasal- and lacrimal arteries of the lids and from the anterior ciliary arteries. The venous drainage from the conjunctiva is to the anterior ciliary vein along with the superior and inferior ophthalmic veins. The conjunctival lymphatic vessels form an irregular mesh under the epithelium and drain to the superficial parotid (pre-auricular), submandibular, and deep cervical lymph nodes (Duke-Elder & Wybar 1961). Microscopically, the normal conjunctiva consists of two histological layers, namely the epithelium and the underlying lamina/substantia propria. The epithelium is mainly composed of stratified columnar epithelium; however, in the transition zones close to the lid margin and the corneal limbus, the conjunctival epithelium transforms to non-keratinizing stratified squamous epithelium. Numerous mucous-secreting goblet cells and melanocytes are present within the epithelium. The lamina propria may be subdivided into two histological layers: a superficial adenoid layer and an underlying fibrous layer. The adenoid layer consists of loose connective tissue that contains lymphocytes, mast cells, dendritic cells, and macrophages. This lymphoid tissue is known as mucosa-associated lymphoid tissue (MALT) (Knop & Knop 2000), in which most conjunctival lymphomas arise (Mikkelsen et al. 2018). The deep fibrous layer consists of dense connective tissue in which collagen and elastic fibres are found. This layer is more properly considered a subconjunctival connective tissue layer, and in it lie the vessels and nerves supplying the conjunctiva (Duke-Elder & Wybar 1961). Cells in our body are derived from one of the three germ layers known as the ectoderm, the endoderm, and the mesoderm. The ectoderm can be further subdivided into the surface ectoderm and the neuroectoderm – the latter giving rise to the neural crest and the neural tube. The conjunctiva is derived from the surface ectoderm, which also gives rise to other mucosal membranes of the head and neck, such as the surface epithelium of the oral and nasal cavities (Duke-Elder & Cook 1963). In the sixth week of gestation, two small folds of surface ectoderm (the frontonasal process and the maxillary process) become visible cranially and caudally to the developing cornea. These two processes give rise to the eyelids, and they grow towards each other to merge in the eighth week of gestation. Between the mesodermal developing cornea and the merged early eyelids, a space is formed called the conjunctival sac. The conjunctival sac is visible from around the tenth week of gestation, and, inside this space, the conjunctival mucous membrane develops. Between the fifth and seventh month of gestation, the eyelids separate again, and the fornical conjunctival folds become visible in the last month of gestation (Duke-Elder & Cook 1963). The function of the conjunctiva is to provide a frictionless surface that allows for free and smooth eye movement. Furthermore, the conjunctiva plays an important role in tear film stability, corneal transparency, and in protecting the eye and orbit from infection (Seregard et al. 2015). Melanocytes are the pigment-producing cells found in the skin, hair, and eyes (Busam et al. 2014). The pigment produced by melanocytes is called melanin, and, in the skin, melanin has great importance in protection from ultraviolet (UV) radiation from the sun by forming an intracellular cap, protecting the nucleus of the surrounding epithelial cells (Cichorek et al. 2013; Busam et al. 2014; Mikkelsen et al. 2016). Melanocytes can also be found in most mucosal membranes, where their role is still not understood (Mikkelsen et al. 2016). Melanocytes develop from melanoblasts that are generally thought to originate in the neural crest, a part of the neuroectoderm (Cichorek et al. 2013). The neural crest harbours multipotent stem cells, which can develop into neurons, Schwann cells, other glial cells, chondrocytes, muscle cells, endocrinal cells, and melanocytes (Hirobe 2011). From the neural crest, melanoblasts migrate dorsolaterally between the ectoderm and the somites to their definitive sites of colonization, following the route of other neural structures, such as peripheral nerves (Thomas & Erickson 2008; Hirobe 2011; Cichorek et al. 2013; Agarwalla et al. 2015). Notably, a subpopulation of melanoblasts follow an alternative ventral route (Cichorek et al. 2013). During the migration from the neural crest, the multipotent melanoblast gradually matures, and in the case of skin melanocytes, the final maturation into melanocytes takes place within the dermis (Hirobe 2011; Cichorek et al. 2013; Busam et al. 2014). This process may be the same in the conjunctiva; however, this is not known. The maturation process is regulated by the transcription factor Sox-10, which activates the microphthalmia transcription factor (MITF), which is the major driver of the melanocytic phenotype (Busam et al. 2014). Interestingly, experiments have shown that terminally differentiated melanocytes are able, under certain conditions, to dedifferentiate, self-renew, and even differentiate to other neural crest-derived cells such as Schwann cells (Dupin et al. 2000). This is thought to be a common feature of neural crest cells, and, seen in this light, it is not surprising that other neural crest-derived tumours, such as schwannomas, may also present a pigmented phenotype (Agarwalla et al. 2015). Microscopically, melanocytes are small round or oval cells with clear cytoplasm in hematoxylin and eosin (H&E)-stained sections. In the skin and conjunctiva, the melanocytes are distributed territorially, with a regular distance within and just beneath the basal epithelial cell layer. Variable numbers of melanocytes are seen in the basal layer of the perilimbal conjunctiva (Spencer 1996). Melanocytes have several dendrites that function as intercellular contacts between the melanocyte and several basal epithelial cells. Ultrastructurally, melanocytes are characterized by a prominent Golgi complex, rough endoplasmatic reticulum, numerous mitochondria, and the presence of melanosomes, the unique ultrastructural hallmark of melanin-producing cells. Melanosomes contain enzymes involved in melanogenesis (Busam et al. 2014). The concept of cancer being a disease of the genome where a single cell overcomes the inherent suppression of cell division due to changes in the chromosomes was first proposed in 1914 by Theodor Boveri (1862–1915) (Manchester 1995). In 1958, Francis Crick (1916–2004) initially described the "central dogma of molecular biology" as the process of the transfer of genetic information from nucleic acid (DNA) to nucleic acid (RNA) to protein, for which he was awarded the Nobel Prize in medicine in 1962 (Crick 1958). Since the early 1970s, molecular oncology research has focused on identifying genes that are frequently mutated in cancer, and it is now generally accepted that cancer arises through the accumulation of mutations in genes (oncogenes) recognized as either proto-oncogenes or tumour suppressor genes. Proto-oncogenes are defined as normal genes that, if overexpressed or constitutively active due to a mutation, can contribute to cancer formation. A myriad of such genes has been identified and includes BRAF, NRAS, and MYC. On the other hand, tumour suppressor genes are genes that protect the cell from becoming cancerous, and when underexpressed or mutated, causing a loss or decrease of function, the cell is likely to progress to cancer. Examples of such genes include NF1, TP53, and PTEN. Recently, a third class named "stability genes" has been identified. Mutations in these genes result in increased mutational rate throughout the genome, which increases the risk of mutations in proto-oncogenes and tumour suppressor genes (Vogelstein & Kinzler 2004). Regardless of the location of genetic alterations in either proto-oncogenes or tumour suppressor genes, the malignant phenotype is characterized by various cellular functions, conceptualized by Hanahan and Weinberg in 2000 as the "hallmarks of cancer" (Hanahan & Weinberg 2000; Hanahan & Weinberg 2011). These hallmarks define eight characteristic features of cancer cells, namely (i) sustained proliferative signalling, (ii) evasion from growth suppressors, (iii) evasion from apoptosis, (iv) limitless replicative potential, (v) sustained angiogenesis, (vi) tissue invasion and metastasis, (vii) altered energy metabolism, and (viii) evasion from immune destruction (Hanahan & Weinberg 2000; Hanahan & Weinberg 2011). In melanoma, genetic alterations disrupt a common set of key signalling pathways, and in particular the mitogen-activated protein kinase (MAPK) pathway is affected in most melanomas at an early stage (Figure 2) (Bastian 2014). The MAPK pathway is activated by mutations at several levels in the pathway. First, activating mutations are found in the upstream surface receptor at the level of KIT. Secondly, mutations are seen in RAS family members acting at the level directly downstream of surface receptors along with negative regulators of RAS (e.g. NF1 and SPRED1). Thirdly, mutations can be found in RAS effectors acting at the levels further downstream (e.g. BRAF, MEK, and cyclin D1) (Figure 2) (Dahl & Guldberg 2007; Zhang et al., 2016; Bastian et al., 2018). In untreated melanomas, alterations of these driver genes tend to be mutually exclusive; however, they can co-occur in melanomas following targeted therapy and contribute to resistance of therapy (Bastian et al., 2018). Following the initial activation of MAPK by early mutations in driver genes in melanoma, a myriad of secondary mutations and alterations occur (Bastian 2014; Shain et al. 2015). A common target for these secondary mutations is the G1/S checkpoint, and mutations affecting this checkpoint allow the cell to override the ability to restrain cell-cycle entry (Bastian 2014). Furthermore, mutations affecting pathways related to the SWI/SNF chromatin remodelling complex are often found, including inactivations of the histone modifier BAP1, which is often mutated in uveal melanoma (Harbour et al. 2010; Smit et al. 2018). Other secondary alterations in melanoma include deletions or mutations in CDKN2A (encoding p16), amplification or mutation of CDK4 (encoding the target kinase of p16), along with loss of RB1. Several of these events can exist as germline alterations predisposing to melanoma. The most frequent mutations in cutaneous melanoma are found in the promotor region of TERT, resulting in immortalization of the cell due to increased expression of telomerase. The mechanisms leading to mutations in driver genes of melanoma vary considerably depending on the type of melanoma (Bastian 2014). The genome of skin melanoma often carries numerous mutations related to ultraviolet light (UV) exposure, mostly cytosine to thymidine (C>T) transitions (Trucco et al. 2019). Melanomas occurring in skin with some degree of cumulative sun damage (CSD) are among the most highly mutated cancers overall, harbouring approximately 30 mutations/Mb of DNA, which is equal to 100 000 mutations pr. genome (Hayward et al. 2017). Skin melanomas with a low degree of CSD (non-CSD) only have about half the mutational load (e.g. 15 mutations/Mb DNA). In contrast, acral melanoma and mucosal melanoma have considerably lower mutational burdens and typically lack a UV signature. Instead, these melanoma genomes are characterized by numerous copy-number variations (CNV) and structural variants (SV). In cutaneous melanoma, atypical mutations in BRAF are found in CSD melanoma, while the BRAF-V600E mutation is associated with non-CSD melanoma and development from an acquired nevus (Shain et al. 2015). Uveal melanoma stands out as a separate melanoma subtype derived from non-epithelial melanocytes resembling melanomas occurring in blue nevi and internal organs, all characterized by Gαq signalling. These genomes are characterized by a lack of highly rearranged genomes along with a low mutational burden, mostly with primary mutations in GNAQ or GNA11 followed by few progression mutations in SF3B1, EIF1AX, and BAP1 (van Poppelen et al., 2018; Shain et al. 2019). Genetics have been shown to play a central role in cancer starting in a normal cell that progresses to a premalignant lesion and further develops into malignancy and metastases. In recent years, we have learned much about the important roles of certain genes and mutations in cancer, but the inadequacy of genetics alone in explaining cancer biology has been known for many years. Gene expression is the process transferring genetic information to a functional gene product, and this process contains many steps, including transcription, RNA splicing, translation, and post-transcriptional modification of the gene product (most often a protein). Several mechanisms contributing to carcinogenesis and cancer biology by altering the gene expression levels have been recognized, including a spectrum of epigenetic mechanisms such as gene-silencing by methylation, modification of chromatin structures, and post-transcriptional gene regulation. Ultimately, disturbances of the gene expression lead to altered signalling of the affected genes, which causes a dysregulation of various signalling pathways. In cancers, such as breast cancer, cutaneousmelanoma, and diffuse large B-cell lymphoma (DLBCL), gene expression profiling has revealed several subtypes that correlate with prognosis, course of disease, and response to certain treatments (Alizadeh et al. 2000; Perou et al. 2000; Mikkelsen et al. 2018). In conjunctival melanoma, no profiling of cancer gene expression has been carried out. However, several studies have investigated the expression of a few or single targets, such as KIT and beta-catenin (Beadling et al. 2008; Reddy et al. 2017). Recently, a sophisticated study from the Netherlands showed that enhancer of zeste homolog 2 (EZH2) was highly expressed in conjunctival melanoma without having concurrent mutations in the gene (Cao et al. 2018). miRNAs are small non-coding RNA molecules (~22 nucleotides) that can regulate gene expression at the post-transcriptional level. Transcription regulation is recognized as an epigenetic event and can be performed by the miRNA according to one of the following mechanisms (Streicher et al. 2012; Fleming et al. 2014): (1) cleavage of the mRNA strand resulting in complete mRNA degradation, (2) shortening of the poly-A-tail resulting in destabilization of the mRNA, or (3) inhibition of ribosomes resulting in less efficient mRNA translation. Mature miRNAs interact with the dicer complex and form the RNA-induced silencing complex (RISC) that mediates the gene silencing functions. The miRNA strand is complementary to a region of the mRNA and it seems that one miRNA may have several target mRNAs. For successful target binding, the seed region (the first 2–7 nucleotides of the miRNA) has to match perfectly with the mRNA. In animals, this target sequence is often at the 3-UTR of the mRNA and the binding of the entire miRNA sequence is often imperfect (Streicher et al. 2012; Fleming et al. 2014). In cancer, upregulation of a miRNA targeting a tumour suppressor gene leads to degradation of the gene transcript, ultimately leading to inhibition of the tumour suppressor gene and promotion of tumour formation. In contrast, downregulation of a miRNA targeting an oncogene leads to decreased degradation of the oncogene transcript, leading to enhanced oncogene expression and increased tumour formation (Streicher et al. 2012; Fleming et al. 2014). However, miRNAs most often have multiple targets accounting for their ability to contribute to several cellular pathways, thus making the exact function of specific miRNAs difficult. In some cancers, such as breast cancer, miRNAs can be used as novel prognostic markers (Zografos et al. 2019). In conjunctival melanoma, a study by our group identified 25 upregulated and 1 downregulated miRNAs in conjunctival melanoma (Larsen et al., 2016a,b). Several of these miRNAs have been previously identified in cutaneous melanoma, and the study provides an entry point for studying miRNA in conjunctival melanoma as potential prognostic and therapeutic markers. The first mention of a conjunctival malignant melanoma entity in the English literature was by the American surgeon Benjamin Travers (1783–1858) in his book A Synopsis of the Diseases of the Eye and their Treatment from 1820, which is also recognized as the first English text book in ophthalmology (Travers 1820). In this work, Travers describes a bizarre case of "a dark purple conjunctival tumour…measuring a quarter of an inch in thickness…on an intact cornea and sclera" (Travers 1820). However, the first truly scientific report of a "melanosarcoma" of the conjunctiva was authored by the German surgeon Paul Baumgarten in 1852 (Baumgarten 1852). In the following decade, numerous similar phenomena were reported by different authors using names like nævo-carcinoma, malignant nevus, melano-carcinoma, epitheliomata, or most frequently sarcomata (Duke-Elder & Leigh 1965). Epithelial sarcoma was the term used by Verhoeff and Loring in 1903, when they published the first review of the literature, including 70 cases (Verhoeff & Loring 1903). In 1937, Algernon B. Reese (1896–1981) used the term "melanoma", and this was probably the first time the term was used for this entity in the English literature (Reese 1937). Sir Stewart Duke-Elder (1898–1978) noted in his Textbook of Ophthalmology from 1940 that most of these entities were probably the same disease, namely malignant melanoma (Duke-Elder 1940). Conjunctival melanoma appeared as a separate entity in the 1952 first edition of the bible of eye pathology, today known as Spencer's Ophthalmic Pathology (1952), and melanoma was also the term used by Lorenz E. Zimmerman (1920–2013) in 1958 when he described late conjunctival melanoma metastases (Lewis & Zimmerman 1957). It is worth noting that Frederick Verhoeff (1874–1968) described local recurrence and metastases as challenging features in conjunctival melanoma in 1903. These are still challenging for ocular oncologists in the modern era of personalized medicine, and molecular biology may help in elucidating perspectives of metastasis that will aid treatment of these patients. The annual incidence of conjunctival melanoma is 0.2–0.8 per million in Caucasian populations (Seregard & Kock 1992; Norregaard et al. 1996; Seregard 1998; Tuomaala et al., 2002a,b; Isager et al. 2005; Missotten et al., 2005; Larsen 2016; Beaudoux et al. 2018; Ghazawi et al. 2019). This is considered a high incidence rate when comparing the surface area to that of the skin and other mucosal membranes. Conjunctival melanoma almost exclusively occurs in Caucasians and is very rare among Blacks and Asians (Hu et al. 2008). In a recent study from South Korea, the incidence has been reported to be 0.12 annual cases per million (Park et al. 2015). During the period 1943–1997, the incidence rate has been reported stable in the Danish population (Isager et al. 2005); however, recent studies from Sweden, Finland, and Denmark all report an increase in incidence in recent years (Tuomaala et al., 2002a,b; Triay et al. 2009; Larsen 2016). In Denmark, this increase is most significant regarding cases located in the sun-exposed conjunctiva, indicating that ultraviolet radiation may play a role in the increase (Larsen et al., 2016a,b). The increase in incidence is also found in skin melanoma but is absent in uveal melanoma (Virgili et al. 2007). The distribution of conjunctival melanoma among males and females is considered to be equal; however, studies from Finland, Canada, and United States have pointed towards a slight male predomination (Tuomaala et al., 2002a,b; Yu et al. 2003; Ghazawi et al. 2019). Furthermore, some studies have reported male patients to be younger at the time of diagnosis (Norregaard et al. 1996; Tuomaala et al., 2002a,b; Missotten et al. 2005; Shields et al. 2011). Most patients presenting with conjunctival melanoma are middle-aged, with a mean age of 55–65 years, and children are only affected casuistically (Norregaard et al. 1996; Tuomaala et al., 2002a,b; Missotten et al. 2005; Tab an & Traboulsi 2007; Hu et al. 2008; Triay et al. 2009). Conjunctival melanoma arises from melanocytes present in the conjunctiva, and there are three recognized pathways leading from a benign melanocyte to a conjunctival melanoma. A conjunctival melanoma can develop from a pre-existing melanocytic neoplasia, such as primary acquired melanosis (PAM), from a benign nevus, or it can develop from a single melanocyte located in the conjunctiva without any present premalignant lesion (de novo). The most common precursor lesion for conjunctival melanoma is PAM, which has been reported to be present in 42–75% of all cases of conjunctival melanoma, most of these occurring in middle-aged Caucasians (Jakobiec et al. 1989a,b; Anastassiou et al. 2002; Tuomaala et al., 2002a,b; Missotten et al. 2005; Shields & Shields 2009; Triay et al. 2009; Larsen et al. 2015). Primary acquired melanosis is a controversial term, as not all PAMs are considered premalignant. Historically, the term PAM included both a clinical and a histopathological phenotype, leading to much confusion. Clinically, PAM is characterized as a unilateral, flat, and variably brown lesion with patches of pigmentation located in the conjunctiva with or without extension to the eyelid skin and/or cornea (Jakobiec et al., 1989a,b; Seregard 1998; Shields & Shields 2009; Larsen 2016). However, histopathologically, PAM can be further divided into PAM with cellular atypia (PAM+), which resembles the premalignant melanoma in situ of skin melanoma to various degrees, and PAM without cellular atypia (PAM−), which is recognized as a truly benign lesion without malignant potential (Jakobiec et al., 1989a,b, Jakobiec et al. 2018). Whether a PAM is classified as PAM+ or PAM− has been associated with some degree of inter- and intra-observer variation, and as an attempt to resolve the confusion and bias, a new histopathological grading system was proposed a few years ago, introducing the term conjunctival intraepithelial melanocytic neoplasia (C-MIN) (Damato & Coupland 2008). In contrast to PAM, the grading of C-MIN uses a formalized score to describe the morphological changes associated with what is clinically seen as a hyperpigmentation of the conjunctiva. The scoring system goes from 1 to 10, where 1 is hypermelanosis without hyperplasia and 10 equals melanoma in situ (Damato & Coupland 2008). Conjunctival intraepithelial melanocytic lesion (CMIL) is the term adopted in the recent WHO classification that suggests a new staging system of neoplastic changes ranging from melanocytic hyperplasia to melanoma in situ (2018). These three staging systems are currently being evaluated by a large international consortium. Even though conjunctival nevi rarely progress into melanoma (Shields & Shields 2004), 2–40% of conjunctival melanomas arise in a pre-existing nevus (Folberg et al. 1989; Anastassiou et al. 2002; Tuomaala et al., 2002a,b; Missotten et al. 2005; Shields et al. 2011; Larsen et al., 2016a,b). Histopathologically, conjunctival nevi are characterized by melanocytes (so-called nevus cells) located outside the epithelium that form characteristic nests (Folberg et al. 1989). Nevi can be further subclassified according to the location of the nevus in the conjunctiva (Folberg et al. 1989; Seregard et al. 2015). Nevi are typically formed in the first decade of life, where the nevus can be found in the junctional zone just beneath the epithelium (junctional nevus) (Folberg et al. 1989; Seregard et al. 2015). With age, the nevus descends into the substantia propria (co