NF1 mutations in conjunctival melanoma

Background Conjunctival melanoma is a potentially deadly eye tumour. Despite effective local therapies, tumour recurrence and metastasis remain frequent. The genetics of conjunctival melanomas remain incompletely understood. Methods A large cohort of 63 conjunctival melanomas was screened for gene mutations known to be important in other melanoma subtypes by targeted next-generation sequencing. Mutation status was correlated with patient prognosis. Results Frequent mutations in genes activating the MAP kinase pathway were identified. NF1 mutations were most frequent (n = 21, 33%). Recurrent activating mutations were also identified in BRAF (n = 16, 25%) and RAS genes (n = 12, 19%; 11 NRAS and 1 KRAS). Conclusions Similar to cutaneous melanomas, conjunctival melanomas can be grouped genetically into four groups: BRAF-mutated, RAS-mutated, NF1-mutated and triple wild-type melanomas. This genetic classification may be useful for assessment of therapeutic options for patients with metastatic conjunctival melanoma

There are two main therapeutic avenues for metastatic melanoma. Firstly, targeted small inhibitors dampening pathologically activated cell-intrinsic signalling mechanisms, with the most effective to date being a combination of BRAF and MEK inhibitors in BRAF-mutated melanoma. 32 Secondly, immunotherapies applying anti-CTLA-4 and anti-PD-1 antibodies have shown impressive response rates in cutaneous and mucosal melanoma. [33][34][35] Both approaches may be clinically useful in advanced conjunctival melanoma. 36 Our study aimed to further elucidate genetic events in conjunctival melanoma by analysing a large tumour cohort with a targeted next-generation sequencing assay covering genes that are recurrently mutated in cutaneous and uveal melanoma.

Sample selection and histopathology
Sixty-seven conjunctival melanoma samples were obtained from the tissue archives of the Departments of Ophthalmology, www.nature.com/bjc Dermatology and Pathology of the University Hospital Essen, and the Department of Ophthalmology, University Hospital Tübingen, Germany. The study was approved by the local ethics committee of the University of Duisburg-Essen.
DNA isolation Formalin-fixed, paraffin-embedded tumour tissues were sectioned, deparaffinised and manually microdissected as previously described. 37 Genomic DNA was isolated using the QIAamp DNA Mini Kit (Qiagen, Hilden, Germany).
Targeted sequencing A custom amplicon-based sequencing panel covering 29 genes known to be mutated in melanoma was used (genes listed in Supplemental Table 1), as previously described. 37 Mean coverage of 2094 reads, with a minimum coverage of 30 reads in >80% of the target loci, was achieved. Four samples were excluded from analysis due to low coverage.
Sequence analysis CLC Cancer Research Workbench from QIAGEN® was used for sequence analysis, as previously reported. 37 Mutations were considered if coverage of the mutation site was ≥30 reads, ≥10 reads reported the mutated variant and the frequency of mutated reads was ≥10%.

Associations of mutation status with clinical and pathological parameters
Associations of mutation status with available clinicopathological parameters (listed in Table 1) were explored. Analyses were performed with IBM SPSS Statistics software (version 20.0; International Business Machines Corp., Armonk NY, USA). A p-value of <0.05 was considered statistically significant.
Additionally, mutations in various genes frequently mutated in cutaneous melanoma were detected. The majority of these mutations were of unknown functional consequences (Supplemental Table 2). While a few GNAQ and GNA11 mutations were identified (Fig. 1, Supplemental Table 2), they presumably represent functionally non-relevant bystander mutations, as none of the identified mutations were the activating R183 or Q209 mutations known to occur in uveal melanomas. 14,15,21 Statistical analysis There were no statistically significant associations between clinicopathological parameters with BRAF, RAS and NF1 mutation status (Table 1).

DISCUSSION
To our knowledge, the present study represents the most detailed analysis of gene mutations in conjunctival melanoma to date.
Activating BRAF mutations were detected in 25% of samples, lying within the range of previous studies reporting 14-50%. [24][25][26][27]38 This variation may be due to sample bias or technical differences. In view of the recent development of effective BRAF and MEK inhibitors, the presence of BRAF V600 mutations in conjunctival melanomas is of considerable therapeutic relevance. 39 In addition to known activating NRAS mutations in 18% (11/63) of tumours, we identified an activating KRAS G12A mutation. Being the first report on these mutations in conjunctival melanoma, this finding is reminiscent of cutaneous melanoma, in which KRAS mutations are rare but occur in a mutually exclusive fashion with NRAS mutations. 11 In the proposed TCGA (The Cancer Genome Atlas) genomic classification of cutaneous melanoma, mutations in all three RAS genes are grouped together as RAS-mutated melanomas.
Our study is the first to identify NF1 as a frequently mutated oncogene (33%) in conjunctival melanoma. NF1 has recently been recognised as the third most commonly mutated gene (after BRAF and RAS) in cutaneous melanoma, activating the MAP kinase pathway. 11 In our conjunctival melanoma cohort, NF1 mutations were also present in samples harbouring activating RAS or BRAF mutations (Fig. 1). This is similar to the situation in cutaneous melanoma where the co-occurrence of NF1 with BRAF, RAS and other mutations is well recognised. 11,12,40 NF1 mutations are particularly frequent in melanoma subtypes rarely harbouring BRAF and NRAS mutations, 8,12,41 including melanomas associated with high sun exposure. 8,12 Ultraviolet exposure is a known pathogenic factor in conjunctival melanoma and could explain the high number of NF1 mutations detected. NF1 mutations have been associated with high tumour mutational load and affected patients have been reported to benefit from anti-PD-1 therapy in cutaneous melanoma. 42 This suggests that NF1 mutation status has potential as a biomarker for immunotherapy in conjunctival melanoma.
In summary, our study identifies a range of mutations in conjunctival melanoma. The distribution of activating mutations, with RAS gene mutations occurring not only in NRAS but also KRAS, and NF1 mutations being frequent in tumours lacking BRAF or RAS mutations, suggests that the proposed genetic classification of cutaneous melanomas into BRAF-mutated, RAS-mutated, NF1-mutated or triple-wild-type tumours is also applicable to conjunctival melanoma.