Conjunctival Melanoma: Genetic and Epigenetic Insights of a Distinct Type of Melanoma

Conjunctival melanoma (CjM) is a rare, primary cancer of the ocular region. Genetic and epigenetic characteristics of conjunctival melanoma have not been completely elucidated yet. Conjunctival melanoma presents similarities with cutaneous melanoma, with substantial differences in the biological behavior. We reviewed the genetic and epigenetic insights of CjM involved in invasion and metastatic spread. CjM is commonly characterized by mutations of v-raf murine sarcoma viral oncogene homolog B1 (BRAF), neurofibromin 1 (NF1) and telomerase reverse transcriptase (TERT), high expression of mammalian target of rapamycin (mTOR) and heat shock protein 90 (HSP90), frequent phosphatase and tensin homolog (PTEN) loss and upregulation of specific miRNAs. These features should identify CjM as a distinct subset of melanoma with its own profile, which is more similar to cutaneous melanoma than mucosal melanoma and remarkably different from uveal melanoma.

This review aims to elucidate in detail the genetic and epigenetic features of CjM involved in invasion and metastatic spread in order to identify potential therapeutic targets for this disease. Moreover, we aim to point out that CjM could be identified as a distinct subset of melanoma with specific genetic and epigenetic alterations that are not completely shared with other types of melanoma, such as cutaneous, mucosal or uveal melanoma ( Figure 2).

Methods
The literature search was performed using electronic databases (Pubmed, Scopus and Web of Science) and selected keywords (such as "conjunctival melanoma", "genetic", "pathway"), linked with the Boolean operator "AND" and "OR". Reference list of the articles was manually screened to find other relevant papers through the snowball search technique. A total of 950 full-length papers, including original researches, case reports and reviews, were identified. All the articles regarding genetic and epigenetic of CjM were considered. Papers dealing with the most important pathways involved in cutaneous, mucosal and uveal melanoma were also selected.

Genetic and Epigenetic Features of Conjunctival Melanoma
CjM has several molecular alterations associated with malignant transformation, invasion and distant spread. RAS-RAF-MEK-ERK is one of the pathways more frequently dysregulated in CjM [28]. This pathway transfers the signal from the plasmatic membrane to the nucleus, activating transcriptional factors and regulating gene expression [47]. In CjM, its activation most commonly depends on BRAF, NRAS or KIT mutations [48]. The frequency of BRAF, NRAS and KIT mutations in CjM is more similar to cutaneous melanoma than uveal/mucosal melanoma [28,29,38,[48][49][50][51].
BRAF-mutant conjunctival melanomas usually occur in young males and present with pigmented cells more frequently than BRAF wild-type conjunctival melanomas [51,56]. Similarly, in cutaneous melanoma BRAF mutations are more predominant among younger patients [57]. Moreover, BRAF-mutant conjunctival melanomas are more common on the bulbar than extrabulbar conjunctiva. This higher incidence of BRAF mutations in the bulbar region of the eye, more exposed to sunlight, identifies UV radiations as a possible risk factor for this disease [31][32][33].
BRAF mutations are not significantly associated with increased recurrence, regional metastases or mortality from CjM, but they are correlated with reduced distant metastases free-survival [33,51].
In vitro, Vemurafenib and Dabrafenib inhibit BRAF-mutant CjM cell lines, similarly to cutaneous melanoma cells [40]. In vivo, several BRAF mutated conjunctival melanomas were effectively treated with BRAF inhibitors in monotherapy or in combination with MEK inhibitors [58][59][60][61][62][63]. Thus, as patients with BRAF-mutant cutaneous melanoma are successfully treated with combined BRAF/MEK inhibitors, it can be hypothesized that CjM patients might also benefit from these agents. In conclusion, we believe that tests for BRAF mutations should be included in the management of CjM and that clinical studies with BRAF and MEK inhibitors are required in this setting [64].

NRAS
The NRAS gene encodes N-ras, a GTPase protein, which is involved in the regulation of cellular division [31]. NRAS mutations have been found in almost 20% of conjunctival melanomas [31,38,40,44,45]. Regarding the other types of melanoma, NRAS mutations have been found in about 20% of cutaneous melanomas, 5-13% of mucosal melanomas and 10% of acral melanomas. NRAS mutations have not been detected in uveal melanomas [65]. It is worth noting that benign cutaneous nevi can harbor NRAS mutations [66,67].

KIT
c-KIT mutations have been detected in almost 2-7% of conjunctival melanomas and they are mutually exclusive with NRAS and BRAF mutations, as in cutaneous melanoma [32,36,38,62]. Similarly, in cutaneous melanomas, the incidence of KIT mutations ranges from 5.1% for non 'sun-exposed' patients to 9.8% for chronically 'sun-exposed' patients. KIT mutations are more frequently detected in mucosal (about 11.5% of cases) and acral (10.8% of cases) melanomas while they have not been reported in uveal melanoma [69]. Overall, KIT mutations show a significant association with older age [70].
It has been reported that different ethnic groups express different rates of mutations: CjM of Chinese people expresses a higher level of KIT mutations (11%), but a lower rate of BRAF mutations (8%) than Caucasian people [50].
It is noteworthy that KIT mutations are not directly correlated with KIT gene copy number or CD117, the KIT gene product, expression. Furthermore, not all KIT regions are sensitive to pharmacological inhibition [71][72][73][74][75]. Consequently, we can assume that not all KIT mutations are drivers in melanomas and are not principal therapeutic targets. Partial responses to KIT inhibitors have been observed in less than 20% of patients with acral, mucosal and chronically sun-damaged melanoma patients, reporting a median overall survival of 46 weeks with imatinib and 7.5 months with dasatinib [74,75].

NF1
The Neurofibromin 1 (NF1) gene (17q11.2) encodes neurofibromin 1, which inhibits the Ras protein through the hydrolysis of Ras-bound GTP [76,77]. NF1 mutations, leading to neurofibromin 1 dysfunction, occur in up to 30% of CjM and can be found simultaneously with BRAF and RAS mutations [35]. This co-occurrence has been also detected in cutaneous melanoma [78][79][80]. On the contrary, the co-mutation of NF1 and KIT has not been demonstrated in CjM, while it has been detected in up to 32% of mucosal melanoma [81].
NF1 mutations are particularly frequent in CjM that have been exposed to UVs, highlighting the possible pathogenetic role of sunlight exposure [79,82,83]. NF1 mutations are associated with sunlight exposure also in cutaneous melanoma and are more frequent in the desmoplastic subtype. It has been demonstrated that cutaneous melanomas with NF1 mutations harbor a higher mutational load. This finding, if confirmed in CjM, could help to identify tumors which are more responsive to immunotherapy ( Figure 3A) [20,84].
Activation of PI3K/AKT/mTOR pathway, confirmed by a high expression of phosphorylated AKT, S6, and 4E-BP1, has been reported in CjM [39]. High expression of mTOR has been found in the cytoplasm of 87% of CjM cells, and its phosphorylated form (Ser2448) in the cytoplasm and nuclei in 75% of cells. S6 and its phosphorylated form (Ser235/236) are expressed in 100% and 75% of CjM cells, respectively. The expression of 4E-BP1 and its phosphorylated form (Thr37/46) is predominantly cytoplasmic [39]. p-AKT Ser473 is mostly represented in the nuclei, while p-AKT Thr308 is both nuclear and cytoplasmic [40].
In cutaneous melanoma, the PI3K-AKT pathway is involved mostly in tumor initiation and resistance to treatments [88]. The association between mTOR nonsynonymous mutations and a short survival has been reported in cutaneous and mucosal melanoma patients [89]. In mTOR mutant cell lines, high levels of phosphorylated S6, AKT and 4E-BP1 have been found. In this context, the inhibition of PI3K/AKT/mTOR pathway exerted an antiproliferative effect [89]. Thus, the predictive value of high levels of AKT, phosphorylated S6 and phosphorylated 4E-BP1 for PI3K/AKT/mTOR inhibition therapy in CjM patients should be investigated in future studies ( Figure 3B).

PTEN
Expression of PTEN, which is an AKT/mTOR pathway inhibitor, is low in CjM [39]. This observation strengthens the hypothesis that the mTOR pathway plays an important role in CjM development [39]. PTEN can be found in different cell compartments such as the cytoplasm and nucleus. The nuclear PTEN plays an oncosuppressive role [90,91]; it is abrogated in neoplastic cells by nuclear-cytoplasmic shuttling. Indeed, the nuclear fraction of PTEN is particularly low in CjM cells [43]. Loss of PTEN has also been observed in about 65% of cutaneous melanomas [92], while the lack of PTEN immunostaining has been reported in only 16% of uveal melanoma [93]. The hypothesis that a low expression of PTEN could allow the response to therapies with mTOR inhibitors in CjM patients should be evaluated in further studies [39].

HSP90
Heat shock protein (HSP)90, a chaperone protein, plays a role in the accurate protein folding and stabilization from stress [94,95]. HSP90 expression is higher in CjM cells than in conjunctival nevi. In particular, HSP90 levels are more elevated in recurrent CjM [43]. This evidence could be particularly important for future therapies targeting specifically HSP90 [96,97]. In cutaneous melanoma HSP90 is also highly expressed, but it does not have a prognostic or predictive value [98]. In uveal melanoma, HSP90 expression has been found, but HSP90 inhibitors did not demonstrate clinical efficacy [99,100].

BCL-2
The B-cell lymphoma 2 (BCL-2) gene family encodes Bcl-2, regulatory proteins which control the mitochondrial response to apoptotic signals to preserve the mitochondrial membrane [101]. Bcl-2 can be considered a marker for melanocytic tumors in the conjunctiva [44]. It has been shown that Bcl-2 levels are remarkably higher in CjM than conjunctival nevi. Surprisingly, there was no significant correlation between Bcl-2 expression and clinical parameters or histopathological characteristics of CjM [43]. In head and neck mucosal melanoma, the high expression of Bcl-2, found in 74% of the cases, predicted a better survival [102]. Differently, in uveal melanoma the expression of protein-interacting protein 3 (BNIP3), which belongs to the Bcl-2 family, has a negative prognostic significance [103]. High levels of Bcl-2 in cutaneous melanoma are associated with aggressive behavior and metastatic spread and seem to predict chemoresistance [104].

TERT
Telomerase reverse transcriptase (TERT) is a catalytic subunit of the telomerase and is activated by AKT. TERT catalyzes the addition of repetitive sequences in the terminal TTAGGG of chromosomes, preventing the degradation of the chromosomal terminations with an increased cellular division rate. This mechanism leads to cellular immortality [105,106]. TERT promoter mutations, which cause an increased TERT expression, are detectable in 32-41% of conjunctival melanomas and in 8% of PAM cases. They have not been detected in conjunctival nevi [41,42]. We can assume that the blockage of telomeric loss consequent to TERT promoter mutations leads to a greater stability of the genome. Indeed, while several conjunctival melanocytic nevi harbor BRAF mutations, TERT promoter mutations are detectable only in melanomas and premalignant lesions (such as PAM with atypia), playing a role in tumor progression. TERT promoter mutations detected in CjM consist of C>T or CC>TT nucleotide changes. These alterations can be considered a typical ultraviolet (UV) effect, suggesting the potential role of UV in inducing genetic alterations involved in the pathogenesis of CjM [107,108]. The occurrence of TERT promoter mutations in CjM is similar to cutaneous melanoma in which TERT mutations can be found in 64-68% of lesions, both in primary and metastases, and are associated with a shorter survival [41,109,110]. To date, no specific prognostic role of TERT promoter mutations has been described in CjM. Acral and mucosal melanomas harbor TERT alterations respectively in up to 41% and 8% of the cases [111,112]. The detection of TERT promoter mutations reveals future therapeutic options for CjM. Thus, reverse transcriptase inhibitors, such as azidothymidine (AZT), may become possible candidates for therapies directed against TERT-promoter mutant conjunctival melanomas ( Figure 4A). Imetelstat (GRN163L) is a telomerase inhibitor, which inhibits TERT promoter activity [113]. Other telomerase inhibitors (e.g., MST-312, TmPyP4, BIBR1532, b-rubromycin, PIPER {N,N-0-bis-[2-(1-piperidino)-ethyl]-3,4,9,10-tetracarboxylic-diimide}) have been also developed and warrant further evaluation in CjM patients [114].

CDKN2A (p16)
Cyclin-dependent kinase-inhibitor 2A (CDKN2A) gene (9p21.3) encodes p16ink4a (p16) protein, which regulates the cell cycle progression through the inactivation of the complex cyclin-dependent kinase 4/6 (CDK4/6)-cyclin D [115]. p16-inactivating mutations cause the loss of its inhibitory function of CDK4/6-cyclin D complex in the G1 to the S phase transition, increasing mitotic activity [115]. CDKN2A mutations can be found both in cutaneous and in CjM [45,116]. Furthermore, CDKN2A germline mutations are associated with familiar melanomas [45,117]. Acral melanoma expresses mutations of the CDK4/6 pathway in about 82.7% of the cases [118]. CDKN2A alterations have been also found in mucosal melanoma of the oral cavity, but they are not related to specific clinicopathological subsets [119]. To date, CDKN2A mutations have never been reported in uveal melanoma [120].
CjM show a lower level of nuclear p16 than benign melanocytic lesions and PAMs with atypia. It has also been highlighted that lesions with thickness lower than 2 mm express higher levels of p16 [45]. In conclusion, we believe that CDKN2A mutations can be of interest as a potential therapeutic target for CjM and can also be useful for the differential diagnosis between CjM and benign atypical conjunctival nevi [45].

Other Genetic Alterations and Chromosome Abnormalities in Conjunctival Melanoma
Other genetic alterations have also been identified in CjM but their role in the pathogenesis of this malignancy and their significance as potential therapeutic target require further clarification.
In primary CjM, CDKN1A (encoding p21) and Runt-related transcription factor 2 (RUNX2) genes, both localized on 6p21.2, are frequently amplified [49]. p21 is a tight-binding inhibitor of CDKs and acts as a regulator of the cell cycle at the G1-S checkpoint [121]. RUNX2 encodes a transcriptional factor that is part of the RUNT family, whose role in the metastatic process has already been investigated [122]. Furthermore, metastatic CjM conjunctival melanoma shows the amplification of MLH1 (3p22.1) and TIMP2 (17q25.3) and the deletion of MGMT (20q26.3) and ECHS1 (10q26.3) [49]. The protein encoded by MLH1 is involved in the DNA repair process [123,124]. TIMP2 encodes for a matrix metalloproteinase that is critical for tissue homeostasis [125]. Deletion of MGMT, which is involved in genome stability, has been detected in many cancer types, including cutaneous melanoma [49]. ECHS1 encodes an enzyme of the fatty acid beta-oxidation, but is also able to interact with STAT3 and has been found altered in many cancer types [28,49,64].
Copy number alterations (CNAs) appear more frequently in BRAF/NRAS wild-type CjM and are principally represented by losses of 1p, 3q, 6q, 8p, 9p, 10, 11q, 12q, 13, 15p and 16q, and gains of 1q, 3p,  6p, 7, 8q, 11q, 12p, 14p and 17q [31,126]. These alterations do not seem to be related to clinical features. The deletion of 10q only was correlated with shorter metastases-free survival, lymphatic invasion and major tumor thickness in 59 CjM patients [126]. Oncosuppressor genes such as NEURL1, PTEN, RASSF4, DMBT1, C10orf90 and C10orf99 are encoded from this region. Moreover, there is a higher frequency of 10q loss in BRAF mutant CjM [126]. In uveal melanoma, typically BRAF-wild type, the most frequent chromosome abnormalities, such as chromosome 3 monosomy and gain of chromosome 8q, demonstrated a prognostic value for relapse, but they did not predict response to treatment [127].
In CjM the frequency of specific chromosomal alterations varies between groups. About 30% of BRAFand 43% of NRAS-mutant conjunctival melanomas show gains of their oncogenic loci. It is possible that a higher expression of oncogenes could play an important role in the tumorigenesis of this malignancy. Gains of 1q, 3p, and 17q occur more frequently in NRAS-mutant than in BRAF-mutant conjunctival melanomas. The loss of chromosome 10 (including PTEN locus) is principally detected in BRAF-mutant CjM. We could assume that tumors with BRAF mutation need a further genetic event, which induces AKT pathway, for their development. On the contrary, this additional event is not necessary for NRAS-mutant CjM, in which the association with chromosome 10 loss is not typical [31].
In the eye, EZH2 protein expression can be detected in the keratinocytes of normal conjunctiva, but it has not been found in normal conjunctival melanocytes and PAM. EZH2 is highly expressed in 50% of primary conjunctival melanomas and 88% of lymph node metastases [46]. High EZH2 is correlated with CjM thickness and poor prognosis. No correlation was found between overexpression of EZH2 and stage, local or distant relapse and tumor localization. In zebrafish xenografts, genetic and pharmacological knockdown of EZH2, through molecules such as GSK503 or UNC1999, reduces tumor growth and colony formation of CjM cells. Inactivation of EZH2 upregulates the oncogene p21/CDKN1A, that controls cellular transition from the G1 to S phase. Moreover, p21 levels are higher after the genetic than the pharmacological inhibition of EZH2, suggesting that EZH2 can regulate transcription using different pathways in addition to its catalytic activity [46]. Inhibition of EZH2 in CjM cells slows the cellular progression to the S-phase and determines cell death through apoptosis and autophagy. Indeed, it increases both the amount of apoptotic cleaved poli-ADP-ribose polymerases (PARPs) and the expression of LC3B-II (Microtubule-associated proteins 1A/1B light chain 3B), a hallmark of autophagy [46].
In conclusion, EZH2 knockdown in CjM cells leads to an S-phase depletion with G1 arrest and accumulation of cells in the G2/M phase. It derives that EZH2 prevents the death of CjM cells [46]. These findings suggest that higher EZH2 is important for tumorigenesis and progression of CjM. For this reason, EZH2 could become a therapeutic target for CjM. EZH2 overexpression has been confirmed in the other types of melanomas associated with metastatic spread and resistance to treatments [128][129][130].
The catalytic activity of EZH2 can be inhibited by some small specific target agents: The phase 1-2 clinical trial (NCT 01897571) with tazemetostat has been designed for patients with advanced solid tumors and B-cell lymphoma [46]. Further investigations are required to define the role of EZH2 in CjM development ( Figure 4B).

miRNA
MicroRNAs (miRNAs) are small, non-coding RNA molecules which work as epigenetic regulators causing post-transcriptional silencing of specific genes through the binding to the 30'UTR of their corresponding mRNAs [131,132]. miRNAs can play the role of oncogenes and oncosuppressors and are involved in the pathogenesis of almost all cancer types [131,133,134]. In CjM, several miRNAs are upregulated and could be considered potential prognostic biomarkers or targets for therapy [33]. Upregulated miRNAs have been also found in cutaneous melanoma [135]. MiR-20b-5p (miR-20b) has been described to be upregulated both in CjM and in cutaneous melanoma. This upregulation is responsible for PTEN suppression [33,136]. MiR-146a acts in the first phases of cancerogenesis in BRAF/NRAS-mutated cutaneous melanoma through NOTCH proteins [137]. The upregulation of miR-146a-5p (miR-146/miR-146a) and miR-146b-5p (miR-146b) has been also reported in CjM [33]. Other miRNAs upregulated both in CjM and cutaneous melanoma are miR-30d-5p (miR-30d), MiR-506-3p (miR-506), miR-509-3p (miR-509) [33,138,139]. The inhibition of miR-509 and miR-506 reduces the capability of CjM cells to grow and invade [35]. The association between the upregulation of mir-3916 and an increased risk of local recurrence of CjM has been pointed out [51]. Some miRNAs are upregulated both in CjM and mucosal melanoma [33]. Other miRNAs are also implicated in uveal melanoma metastatic spread [140].

Conclusions
Overall, CjM is commonly characterized by mutations of BRAF, NF1 and TERT, high expression of mTOR and HSP90, frequent PTEN loss and upregulation of specific miRNAs. These alterations represent potential therapeutic targets. In particular, it could be useful to test the BRAF mutational status considering the high rate of mutations. The anti-BRAF and anti-MEK combination could be a therapeutic option in case of BRAF mutations [58].
Based on the genetic features, CjM can be considered more similar to cutaneous than mucosal melanoma and remarkably different from uveal melanoma. Consequently, we can assume that CjM is a distinct type of melanoma.
Being ocular melanomas, both CjM and uveal melanoma are often excluded from clinical trials, despite their diversity. However, taking into account the genetic profile of CjM and its similarities with cutaneous melanoma, the extension to CjM of the studies proposed for cutaneous melanoma could be encouraged.
While the knowledge of biology has improved over the last years, further information regarding genetic and epigenetic features of CjM is required to address the best targeted treatments. The challenge for the future is the identification of the driver molecular alterations to achieve a clinically relevant therapeutic effect in CjM patients.