Article Text

Genetic landscape and prognosis of conjunctival melanoma in Chinese patients
  1. Hanhan Shi1,2,
  2. Hao Tian1,2,
  3. Tianyu Zhu1,2,
  4. Jie Chen1,2,
  5. Shichong Jia3,
  6. Chunyan Zong1,2,
  7. Qili Liao1,2,
  8. Jing Ruan1,2,
  9. Shengfang Ge1,2,
  10. Yamin Rao4,
  11. Mei Dong5,
  12. Renbing Jia1,2,
  13. Yimin Li1,2,
  14. Shiqiong Xu1,2,
  15. Xianqun Fan1,2
  1. 1Department of Ophthalmology, Shanghai Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China
  2. 2Shanghai Key Laboratory of Orbital Diseases and Ocular Oncology, Shanghai, China
  3. 3Tianjin Eye Hospital, Tianjin Key Lab of Ophthalmology and Visual Science, Nankai University Affiliated Eye Hospital, Tianjin Eye Institute, Tianjin, China
  4. 4Department of Pathology, Shanghai Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China
  5. 5The Core Laboratory in Medical Center of Clinical Research, Shanghai Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China
  1. Correspondence to Professor Xianqun Fan, Department of Ophthalmology, Shanghai Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China; fanxq{at}sjtu.edu.cn; Dr Shiqiong Xu; 115033{at}sh9hospital.org.cn; Dr Yimin Li; eeminlee{at}163.com

Abstract

Aims Conjunctival melanoma (CoM) is a rare but highly lethal ocular melanoma and there is limited understanding of its genetic background. To update the genetic landscape of CoM, whole-exome sequencing (WES) and targeted next-generation sequencing (NGS) were performed.

Methods Among 30 patients who were diagnosed and treated at Shanghai Ninth People’s Hospital, Shanghai Jiao Tong University School of Medicine, from January 2018 to January 2023, WES was performed on 16 patients, while targeted NGS was conducted on 14 patients. Samples were analysed to identify the mutated genes, and the potential predictive factors for progression-free survival were evaluated. Furthermore, the expression of the mutated gene was detected and validated in a 30-patient cohort by immunofluorescence.

Results Mutations were verified in classic genes, such as BRAF (n=9), NRAS (n=5) and NF1 (n=6). Mutated FAT4 and BRAF were associated with an increased risk for the progression of CoM. Moreover, decreased expression of FAT4 was detected in CoM patients with a worse prognosis.

Conclusions The molecular landscape of CoM in Chinese patients was updated with new findings. A relatively high frequency of mutated FAT4 was determined in Chinese CoM patients, and decreased expression of FAT4 was found in patients with worse prognoses. In addition, both BRAF mutations and FAT4 mutations could serve as predictive factors for CoM patients.

  • Conjunctiva
  • Genetics
  • Neoplasia

Data availability statement

Data are available on reasonable request.

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This is an open access article distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited, appropriate credit is given, any changes made indicated, and the use is non-commercial. See: http://creativecommons.org/licenses/by-nc/4.0/.

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WHAT IS ALREADY KNOWN ON THIS TOPIC

  • The molecular characteristics of conjunctival melanoma (CoM) in Caucasian patients have been previously elucidated. However, there is a lack of comprehensive description regarding the molecular features in Asian patients.

WHAT THIS STUDY ADDS

  • We have updated the molecular landscape of CoM in Chinese patients. Additionally, a relatively high frequency of FAT4 mutations was observed among Chinese CoM patients, and decreased expression of FAT4 was identified in patients with poorer prognoses.

HOW THIS STUDY MIGHT AFFECT RESEARCH, PRACTICE OR POLICY

  • FAT4 mutation was related to a higher risk for CoM progression, indicating that FAT4 is a novel predictive factor and therapeutic target. Medical professionals should increase their focus on patients with CoM who have an FAT4 mutation.

Introduction

Conjunctival melanoma (CoM), accounting for 5%–7% of all ocular melanomas, is a rare but potentially deadly disease.1 According to previously published case studies, the 5-year mortality rate is 5%–30.5%, and the 10-year mortality rate is 14%–59%.2–4 The most common treatment is wide excision with cryotherapy, and exenteration is a reasonable option when advanced disease is present.5 However, despite various therapies, the recurrence, metastasis and mortality rates among CoM patients remain high. Insight into the molecular characteristics of CoM enhances the development of targeted therapies and offers the possibility of improving disease control and prolonging the survival of patients with distant metastases.

Previous studies revealed the molecular characteristics of CoM in Caucasian patients.6–8 The genomic characteristics of CoM were well defined by the classification of cutaneous melanoma (CM) proposed by TCGA, with the presence of four typical subclasses defined on the basis of the most frequently mutated genes: BRAF, RAS, NF1 and triple wild-type.8 A high frequency of BRAF mutations was found (14%–50% of patients with CoM).9–11 Other mutations in genes such as NRAS, NF1, KIT and TERT were also identified.6 Recently, several studies have demonstrated that a greater understanding of the molecular features of CoM may improve clinical management. In large tumours carrying the BRAF V600E mutation, neoadjuvant therapy with combined systemic BRAF and MEK inhibitors followed by local excision may be used as an alternative to exenteration.12 Meanwhile, the importance of molecular-targeted inhibitors for treating locally advanced and metastatic BRAF-mutated CoM was reported.13 14 Generating a landscape of the genetic background of CoM is important for guiding treatment and performing large clinical trials. However, the molecular characteristics of Asian patients remain poorly described.

In this study, we assessed the molecular landscape of CoM through whole-exome sequencing (WES) or targeted next-generation sequencing (NGS). A relatively high frequency of mutated FAT4 was determined in Chinese CoM patients, and decreased expression of FAT4 was found in patients with worse prognoses. In addition, FAT4 and BRAF mutations could serve as prognostic factors.

Methods

Patients

Thirty consecutive patients with CoM treated at Shanghai Ninth People’s Hospital, Shanghai Jiao Tong University School of Medicine, from January 2018 to January 2023, were included. The diagnoses were based on the clinical presentations and confirmed by pathological reports. The exclusion criteria were as follows: (1) insufficient tissue for testing; (2) inadequate follow-up period (under 3 months); (3) incomplete data collection and (4) patients’ unwillingness to undergo DNA sequencing. The patients underwent imaging examinations including CT and ultrasound during regular follow-up. Disease progression was defined as pathology-proven recurrence or pathology/imageology-proven metastasis. Specifically, recurrence was defined as pathology-proven ocular recurrence, and pathology/imageology-proven metastasis included pathology-proven local metastasis and imageology-proven distant metastasis.

Tumour samples and DNA sequencing

Fresh frozen tumour tissues and matching blood samples (n=30) for DNA sequencing were obtained from the biospecimen bank of Ninth People’s Hospital, Shanghai Jiao Tong University School of Medicine, with written informed patient consent. With the patients’ willingness and written consent, tumour DNA was extracted from 16 CoM patients and prepared for WES; targeted NGS covering 159 genes was performed on the samples from the remaining 14 patients (online supplemental table 1). Germline DNA mutations were extracted from matched blood samples to identify somatic variations found only in tumour samples. WES was carried out using a TWIST Comprehensive Exon kit (102033, Twist Bioscience) and an Illumina NovaSeq 6000, with sequencing performed at a depth of 60X. Clean reads were aligned against the human reference genome hs37d5 (based on GRCh37 assembly with human virus sequences) with default parameters. Mutect2 in GATK3.715 was used to detect possible single nucleotide variants (SNVs) and small insertions or deletions in the tumour genome. Significantly mutated genes, regarded as candidates for driver genes, were identified via the MutSigCV tool using mutations in all samples as the input and comparing them against the background mutation rate.16

Supplemental material

Immunofluorescence

The tumour tissues were fixed with 4% formaldehyde (I28800, Thermo Scientific) for 15 min and blocked with 5% normal goat serum (005-000-121, Jackson ImmunoResearch) with 0.1% Triton X-100 (A600198-0500, Sangon Biotech) in PBS for 60 min at room temperature. Immunostaining was performed using the FAT4 antibody (PA5-116735, Invitrogen, 1:100 dilution) and Alexa Fluor 488 secondary antibody (A-11034, Invitrogen, 1:1000 dilution). Nuclei were counterstained with 4',6-diamidino-2-phenylindole (DAPI). Images were taken with a ZEISS Axio Scope A1 Upright Microscope.

Statistical analysis

The data were analysed by using SPSS software (V.22.0; IBM) and are reported as the mean±SD or n (%). Univariable Cox regression models were used to identify the relationship between mutations and prognosis. Kaplan-Meier survival curves were applied to evaluate progression-free survival. The log-rank test was used to determine whether there was statistical significance. Disease recurrence and metastasis were considered progression-free survival endpoints. All tests were two sided, and a p<0.05 was considered statistically significant.

Results

Clinical features

Between January 2018 and January 2023, 36 CoM patients underwent resection with or without cryotherapy at our centre. The surgical approaches and procedures were determined based on tumour size, location and partly on the preference of the surgeon. Whenever feasible, surgical tumour-free margins of at least 1 cm were achieved. Three patients were excluded due to unwillingness to undergo DNA sequencing, and three patients were excluded due to insufficient tissue for testing. Overall, 30 patients were included in this study. Based on the patients’ willingness and written informed consent, WES was conducted on 16 patients and targeted NGS was conducted on 14 patients. The patients’ clinical and pathological characteristics as well as the margin status are summarised in table 1 . According to the AJCC eighth edition, 4 (13%) tumours were defined as cT1, 10 (33%) tumours were defined as cT2 and 16 (54%) tumours were staged cT3. No cases of stage cT4 tumours were present.

Table 1

Patient and tumour characteristics

Genetic characteristics of CoM patients

A waterfall plot was constructed to identify the harmful mutations in all 30 CoM patient samples (figure 1). According to the CoM subtypes proposed by The Cancer Genome Atlas (TCGA) and a previous report, the most frequently mutated genes were organised into the following five classifications: CM drivers, uveal melanoma (UM) drivers, cancer-associated drivers, epigenetic drivers and immunity-associated drivers.6

Figure 1

Waterfall plot of 30 CoM patients. CoM, conjunctival melanoma; SNV, single nucleotide variants.

In the CM driver classification, BRAF V600E was the most frequent mutation found in this study and was present in 9 (30%) patients, consistent with previous CoM reports17 18 (online supplemental table 2). NRAS Q61R (n=3), NRAS G13R (n=1) and NRAS G12A (n=1) mutations were also frequent. NRAS Q61R, NRAS G13R and NRAS G12A are located in the Ras domain, a mutation hotspot of the NRAS gene. Moreover, mutated BRAF and mutated NRAS were mutually excluded, and no patient in our study had both mutated BRAF and mutated NRAS, consistent with a previous report.11 We also identified patients harbouring a mutation in the tumour suppressor NF1 (n=6). Mutations in KIT (n=2), a receptor tyrosine kinase that encodes the c-KIT receptor protein, were also found.19 While the mutations we reported in BRAF, NRAS and KIT were all non-synonymous SNVs, the mutations in NF1 were splicing (n=2), non-frameshift deletion (n=2), non-synonymous SNVs (n=1) and stop-gain (n=1) mutations.

In the UM driver category, BAP1 C91Y was detected in one patient, and SF3B1 R625C and SF3B1 R625H were found in three patients. Both mutations in SF3B1 occurred in the hotspot region of the gene. However, no mutations were detected in GNAQ and GNA11, the other two classic mutations in UM.

Mutations were also found in several classical cancer-associated genes, including FAT4 (n=5), TERT (n=3), PTEN (n=3), CSMD1 (n=3), APC (n=3) and TP53 (n=2). In addition to the aforementioned mutated genes, mutations were also identified in ARID1A, FLT1, CDH8, CDH11, SYK, ROS1, ALK, CDKN2A, ATM, SNAI1 and GCSH.

Mutations in epigenetic-related genes play roles in the tumourigenesis of CoM and have been detected in several patients, including KMT2D (n=4), KMT2C (n=3), KMT2B (n=2) and BRD4 (n=1). In the immunity-associated gene category, mutations in MALT1, SHSB3, NOTCH3, RALA, SMC3, IL7R and NOTCH2 were identified. To identify the relationship between genetic background and pathological characteristics, detailed pathological information is displayed in online supplemental figure 1.

The FAT4 gene is a site of potentially critical mutations

Previous studies reported that FAT4 mutations were frequently found in CM cell lines and CoM patients.8 20 High-frequency mutations in FAT4 were previously unreported in Chinese CoM but identified in five patients in this study. FAT4 E1907K, FAT4 E2511K, FAT4 P2547S, FAT4 S3071F and FAT4 P4377S were all detected, three of which are in the same exon region (online supplemental table 2).

Furthermore, the change from glutamic acid to diamino caproic acid altered an acidic amino acid to a basic amino acid, potentially affecting the protein structure and function. Moreover, according to PolyPhen-2 prediction, all the mutations in FAT4 in CoM patients in our cohort were likely to be damaging, with scores of 0.953, 0.977, 0.775, 0.996 and 1.000.

To further validate the function of FAT4, we evaluated its expression in another CoM cohort (n=30). The patients’ clinical and pathological characteristics are summarised in online supplemental table 3. We measured the expression of FAT4 in CoM samples from progression-free and progression-stage patients. The expression of FAT4 in progression-free CoM tissues was significantly higher than that in the other tissues (p=0.05). Decreased expression of FAT4 was found to be related to worse prognoses (figure 2).

Figure 2

Expression of FAT4 in primary CoMs and progressive CoMs. CoM, conjunctival melanoma; DAPI, 4',6-diamidino-2-phenylindole.

FAT4 mutations and BRAF mutations predict poorer prognosis of CoM patients

To evaluate the importance of different mutations in CoM, we analysed the value of several gene mutations as potential predictive factors for progression-free survival in this study (table 2). FAT4 mutations were related to a higher risk for progression (HR 3.912, 95% Cl 1.156 to 13.238, p=0.046). Meanwhile, BRAF mutation was associated with an increased risk for CoM progression (HR 12.505, 95% Cl 3.235 to 48.333, p<0.001). No significant association was found between the other test mutations and worse prognosis in our study. Furthermore, both mutated BRAF and FAT4 predicted a lower progression-free survival probability (log-rank test, p=0.05) (figure 3).

Table 2

Univariate analyses of mutated genes predicting progression-free in CoM patients

Figure 3

Progression-free survival based on FAT4 mutations (A) BRAF mutations (B) NRAS mutations (C) NF1 mutations (D) KIT mutations (E) and TERT mutations (F).

To further evaluate the importance of BARF and FAT4 mutations as potential predictive factors for progression-free survival, separate analyses for recurrence and metastasis were conducted. The results demonstrated that mutated BRAF and FAT4 predicted a higher metastasis probability (log-rank test, p<0.05), and mutated BRAF predicted a higher recurrence probability (log-rank test, p<0.05). The trend was also observed in mutated FAT4 predicting recurrence, without statistical significance because of the limited patient number (online supplemental figure 2). However, the trend was not observed in the other test mutations in our study. Considering that two of the five FAT4 mutation cases had a concurrent BRAF canonical V600E mutation, to clarify the survival function of FAT4 mutation, we performed a stratification analysis by controlling each mutation (online supplemental figure 3). The results showed that FAT4 mutation was associated with rapid progression regardless of the BRAF mutation status (BRAFwt log-rank p=0.038, BRAFmut log-rank p=0.032). Similarly, BRAF mutation was related to a higher risk for progression wherever FAT4 was mutated (FAT4wt log-rank p<0.001, FAT4mut log-rank p=0.039). Moreover, we also evaluated the association between mutated FAT4 and tumour mutation frequency. There was no evidence to suggest that FAT4 mutations were associated with an elevated tumour mutation frequency (online supplemental figure 4).

Discussion

In this study, we summarised the molecular landscape of CoM patients in China through WES and targeted NGS. Based on the detection of FAT4 mutations in CoM patients and PolyPhen-2 prediction, we propose that FAT4 is a potential critical site of mutation in CoM. We confirmed that decreased expression of FAT4 was related to worse CoM patient prognosis in a validation cohort. Based on the updated genetic landscape of CoM, we further explored whether mutated BRAF and FAT4 predicted a worse prognosis.

Our study included more CoM patients with cT2 and cT3 stage tumours than in previously published reports. Some patients have been in part presented in our previous studies.21 This clinical stage distribution was in accordance with a cohort previously published in China.4 De novo tumour origin, non-bulbar location, imbalanced medical care in rural areas and delayed diagnosis in primary care all result in a higher proportion of progressive-stage CoM. Moreover, the different frequencies of mutations in certain genes and several mutations in tumour suppressor genes are also associated with advanced CoM.

Our results revealed that mutations in BRAF and NRAS were predominant in CoM patients in China. Previous studies in Western countries demonstrated that the frequency of BRAF mutations was 14%–50% and that of NRAS mutations was 18%.9–11 The frequencies of BRAF and NRAS mutations were 30% and 17% in China, respectively, consistent with previous reports from Western countries. Mutations in the tumour suppressor gene NF1 were more common in CoM patients than in CM, UM, and mucosal melanoma patients. NF1 mutations were detected in 33% of Caucasian CoM patients.22 23 However, 20% of the patients in our study were confirmed to have NF1 mutations. The patient genetic background and small sample size in our study could explain the different mutation frequencies. In addition, these mutations are potential therapeutic targets. Mutations in BRAF, NRAS, and NF1 activate the Ras-Raf-MEK-ERK (MAPK) pathway and lead to melanoma tumourigenesis.24 Mitogen-activated protein kinase (MEK) inhibitors can pharmacologically suppress the MAPK pathway and provide targeted therapy to patients with mutations.25

Genetically, CoM differs dramatically from UM, the most common ocular melanoma. Important mutations in UM that contribute to the progression of tumours occur in BAP1, SF3B1, GNAQ and GNA11.26 Mutations in BAP1 are associated with the worst prognosis, and immunohistochemistry is used to assess BAP1 expression to predict the prognosis of UM patients.27 28 Even so, analysis of TCGA data demonstrated that the relationship between prognosis and BAP1 mRNA expression is opposite in UM versus CM, indicating the different roles of BAP1 mutations in individual melanomas.29 In our study, the BAP1 C91Y mutation was detected in one patient, who experienced neither recurrence nor metastasis during the follow-up. However, with the small number of tumour samples, we could not draw any conclusions regarding BAP1; additionally, GNAQ and GNA11, the other critical regulators of UM, were not found among our patients.

In addition, we identified frequently mutated FAT4 in CoM patients in China. FAT4, a member of the cadherin family and the upstream transmembrane receptor of the Hippo signalling pathway, plays a critical role in cell proliferation, apoptosis and tumourigenesis.30 31 Low expression of FAT4 has been reported in various tumours, including CM, oesophageal cancer, gastric cancer and colorectal cancer, resulting from mutation, deletion or hypermethylation.32–34 In our study, we found FAT4 E1907K, FAT4 E2511K, FAT4 P2547S, FAT4 S3071F and FAT4 P4377S mutations in CoM patients. All cases in our cohort presented G:C→A:T base changes, the canonical UV mutation signature. Interestingly, in a previously reported sequencing data from patients with CoM, FAT4 mutations were also observed in 9 individuals among 14 CoM patients, exhibiting the same mutation pattern (G:C→A:T base changes) as that identified in our cohort.8 The study updated the molecular landscape of CoM in Caucasian patients, which was validated by our cohort in Chinese patients, indicating that FAT4 is a novel predictive factor and therapeutic target.

To further explore the potential tumourigenesis function of FAT4 in CoM, we validated the expression of FAT4 and found that decreased expression of FAT4 was associated with poor prognosis. Kaplan-Meier survival analysis and further stratification analysis showed that FAT4 mutation was related to a higher risk for CoM progression, indicating that FAT4 is a novel predictive factor and therapeutic target that remains to be further explored.

Limitation

In this single-centre observation cohort study, two sources of bias could not be ignored, including the single centre (restricted sample size and selection bias) and Han People (representing only one part of the wider population). Moreover, a limited sample size influenced the power of the statistical analysis.

Conclusions

In this study, we employed WES and targeted NGS to update the molecular landscape of CoM in Chinese patients. Our findings revealed a relatively high frequency mutated FAT4 among Chinese CoM patients and proposed that FAT4 may serve as a novel mutation site associated with disease progression in CoM.

Supplemental material

Data availability statement

Data are available on reasonable request.

Ethics statements

Patient consent for publication

Ethics approval

This study involves human participants and the study has been approved by the Ethics Committee of Shanghai Jiaotong University (SH9H-2019-T185-2). Participants gave informed consent to participate in the study before taking part.

Acknowledgments

We thank to all conjunctival melanoma patients enrolled in our study and wish them a good health. We also wish to acknowledge Dr Gongwei Long for his help in figure editing.

References

Supplementary materials

  • Supplementary Data

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Footnotes

  • HS, HT and TZ contributed equally.

  • Contributors XF, SX, YL and RJ conceived this project and supervised all the experiments. HS, HT, JC and SJ collected the clinical samples, performed the experiments and drafted the manuscript. TZ, CZ and MD analysed the data. YR and QL was responsible for IF validation. JR and SG revised the manuscript. XF is guarantor.

  • Funding This work was supported by grants from the National Natural Science Foundation of China (82073889, 82303106, U23A20466), the Clinical Research Plan of SHDC (SHDC2020CR1009A), the Science and Technology Commission of Shanghai (22Y31900700, 2022YQ001 and 20DZ2270800), Innovative research team of high-level local universities in Shanghai (SSMU-ZDCX20180401), and Shanghai Ninth People's Hospital, Shanghai University School of Medicine (JYJC202303).

  • Competing interests None declared.

  • Provenance and peer review Not commissioned; externally peer reviewed.

  • Supplemental material This content has been supplied by the author(s). It has not been vetted by BMJ Publishing Group Limited (BMJ) and may not have been peer-reviewed. Any opinions or recommendations discussed are solely those of the author(s) and are not endorsed by BMJ. BMJ disclaims all liability and responsibility arising from any reliance placed on the content. Where the content includes any translated material, BMJ does not warrant the accuracy and reliability of the translations (including but not limited to local regulations, clinical guidelines, terminology, drug names and drug dosages), and is not responsible for any error and/or omissions arising from translation and adaptation or otherwise.