Ocular melanoma consists of posterior uveal melanoma, iris melanoma and conjunctival melanoma. These malignancies derive from melanocytes in the uveal tract or conjunctiva. The genetic profiles of ...these different entities differ from each other. In uveal melanoma,
and
gene mutations are frequently found and prognosis is based on mutation status of
,
and
genes. Iris melanoma, also originating from the uvea, has similarities to the genetic makeups of both posterior uveal melanoma (UM) and conjunctival melanoma since mutations in
and
are less common and genes involved in conjunctival melanoma such as
have been described. The genetic spectrum of conjunctival melanoma, however, includes frequent mutations in the
,
and
promoter genes, which are found in cutaneous melanoma as well. The
status of the tumor is not correlated to prognosis, whereas the
promoter gene mutations are. Clinical presentation, histopathological characteristics and copy number alterations are associated with survival in ocular melanoma. Tissue material is needed to classify ocular melanoma in the different subgroups, which creates a need for the use of noninvasive techniques to prognosticate patients who underwent eye preserving treatment.
The aim of this study was exploration of the genetic background of conjunctival melanoma (CM) and correlation with recurrent and metastatic disease. Twenty-eight CM from the Rotterdam Ocular Melanoma ...Study group were collected and DNA was isolated from the formalin-fixed paraffin embedded tissue. Targeted next-generation sequencing was performed using a panel covering GNAQ, GNA11, EIF1AX, BAP1, BRAF, NRAS, c-KIT, PTEN, SF3B1, and TERT genes. Recurrences and metastasis were present in eight (29%) and nine (32%) CM cases, respectively. TERT promoter mutations were most common (54%), but BRAF (46%), NRAS (21%), BAP1 (18%), PTEN (14%), c-KIT (7%), and SF3B1 (4%) mutations were also observed. No mutations in GNAQ, GNA11, and EIF1AX were found. None of the mutations was significantly associated with recurrent disease. Presence of a TERT promoter mutation was associated with metastatic disease (p-value = 0.008). Based on our molecular findings, CM comprises a separate entity within melanoma, although there are overlapping molecular features with uveal melanoma, such as the presence of BAP1 and SF3B1 mutations. This warrants careful interpretation of molecular data, in the light of clinical findings. About three quarter of CM contain drug-targetable mutations, and TERT promoter mutations are correlated to metastatic disease in CM.
Germline pathogenic variants in the BRCA1-associated protein-1 (
) gene cause the BAP1-tumor predisposition syndrome (BAP1-TPDS, OMIM 614327). BAP1-TPDS is associated with an increased risk of ...developing uveal melanoma (UM), cutaneous melanoma (CM), malignant mesothelioma (MMe), renal cell carcinoma (RCC), meningioma, cholangiocarcinoma, multiple non-melanoma skin cancers, and
-inactivated nevi. Because of this increased risk, it is important to identify patients with BAP1-TPDS. The associated tumors are treated by different medical disciplines, emphasizing the need for generally applicable guidelines for initiating genetic analysis. In this study, we describe the path to identification of BAP1-TPDS in 21 probands found in the Netherlands and the family history at the time of presentation. We report two cases of de novo
germline mutations (2/21, 9.5%). Findings of this study combined with previously published literature, led to a proposal of guidelines for genetic referral. We recommend genetic analysis in patients with ≥2 BAP1-TPDS-associated tumors in their medical history and/or family history. We also propose to test germline
in patients diagnosed with UM <40 years, CM <18 years, MMe <50 years, or RCC <46 years. Furthermore, other candidate susceptibility genes for tumor types associated with BAP1-TPDS are discussed, which can be included in gene panels when testing patients.
Uveal melanoma is a highly aggressive cancer of the eye, in which nearly 50% of the patients die from metastasis. It is the most common type of primary eye cancer in adults. Chromosome and mutation ...status have been shown to correlate with the disease-free survival. Loss of chromosome 3 and inactivating mutations in BAP1, which is located on chromosome 3, are strongly associated with ‘high-risk' tumors that metastasize early. Other genes often involved in uveal melanoma are SF3B1 and EIF1AX, which are found to be mutated in intermediate- and low-risk tumors, respectively. To obtain genetic information of all genes in one test, we developed a targeted sequencing method that can detect mutations in uveal melanoma genes and chromosomal anomalies in chromosome 1, 3, and 8. With as little as 10 ng DNA, we obtained enough coverage on all genes to detect mutations, such as substitutions, deletions, and insertions. These results were validated with Sanger sequencing in 28 samples. In >90% of the cases, the BAP1 mutation status corresponded to the BAP1 immunohistochemistry. The results obtained in the Ion Torrent single-nucleotide polymorphism assay were confirmed with several other techniques, such as fluorescence in situ hybridization, multiplex ligation-dependent probe amplification, and Illumina SNP array. By validating our assay in 27 formalin-fixed paraffin-embedded and 43 fresh uveal melanomas, we show that mutations and chromosome status can reliably be obtained using targeted next-generation sequencing. Implementing this technique as a diagnostic pathology application for uveal melanoma will allow prediction of the patients' metastatic risk and potentially assess eligibility for new therapies.
To explore the genetic background of choroidal and ciliary body melanoma among children and young adults, with special focus on BAP1 germline variants in this age group.
Patients under the age of 25 ...and with confirmed choroidal or ciliary body melanoma were included in this retrospective, multicenter observational study. Nuclear BAP1 immunopositivity was used to evaluate the presence of functional BAP1 in the tumor. Next-generation sequencing using Ion Torrent platform was used to determine pathogenic variants of BAP1, EIF1AX, SF3B1, GNAQ and GNA11 and chromosome 3 status in the tumor or in DNA extracted from blood or saliva. Survival was analyzed using Kaplan-Meier estimates.
The mean age at diagnosis was 17 years (range 5.0-24.8). A germline BAP1 pathogenic variant was identified in an 18-year-old patient, and a somatic variant, based mainly on immunohistochemistry, in 13 (42%) of 31 available specimens. One tumor had a somatic SF3B1 pathogenic variant. Disomy 3 and the absence of a BAP1 pathogenic variant in the tumor predicted the longest metastasis-free survival. Males showed longer metastasis-free survival than females (P = 0.018).
We did not find a stronger-than-average BAP1 germline predisposition for choroidal and ciliary body melanoma among children and young adults compared to adults. Males had a more favorable survival and disomy 3, and the absence of a BAP1 mutation in the tumor tissue predicted the most favorable metastasis-free survival. A BAP1 germline pathogenic variant was identified in one patient (1%), and a somatic variant based mainly on immunohistochemistry in 13 (42%).
Uveal melanoma (UM) has a high propensity to metastasize. Prognosis is associated with specific driver mutations and copy number variations (CNVs), but limited primary tumor tissue is available for ...molecular characterization due to eye-sparing irradiation treatment. This study aimed to assess the rise in circulating tumor DNA (ctDNA) levels in UM and evaluate its efficacy for CNV-profiling of patients with UM.
In a pilot study, we assessed ctDNA levels in the blood of patients with UM (n = 18) at various time points, including the time of diagnosis (n = 13), during fractionated stereotactic radiotherapy (fSRT) treatment (n = 6), and upon detection of metastatic disease (n = 13). Shallow whole-genome sequencing (sWGS) combined with in silico size-selection was used to identify prognostically relevant CNVs in patients with UM (n = 26) from peripheral blood retrieved at the time of diagnosis (n = 9), during fSRT (n = 5), during post-treatment follow-up (n = 4), metastasis detection (n = 6), and metastasis follow-up (n = 4).
A total of 34 patients had blood analyzed for ctDNA detection (n = 18) and/or CNV analysis (n = 26) at various time points. At the time of diagnosis, 5 of 13 patients (38%) had detectable ctDNA (median = 0 copies/mL). Upon detection of metastatic disease, ctDNA was detected in 10 of 13 patients (77%) and showed increased ctDNA levels (median = 24 copies/mL, P < 0.01). Among the six patients analyzed during fSRT, three (50%) patients had detectable ctDNA at baseline and three of six (50%) patients had undetectable levels of ctDNA. During the fSRT regimen, ctDNA levels remained unchanged (P > 0.05). The ctDNA fractions were undetectable to low in localized disease, and sWGS did not elucidate chromosome 3 status from blood samples. However, in 7 of 10 (70%) patients with metastases, the detection of chromosome 3 loss corresponded to the high metastatic-risk class.
The rise in ctDNA levels observed in patients with UM harboring metastases suggests its potential utility for CNV profiling. These findings highlight the potential of using ctDNA for metastasis detection and patient inclusion in therapeutic studies targeting metastatic UM.
IMPORTANCE: It is necessary to understand the mechanisms of metastasis of uveal melanoma to advise patients and develop treatments for this tumor. OBJECTIVE: To examine the stochastic properties of ...primary uveal melanoma including the mutation rate as a function of tumor size and metastatic rate relative to the type of mutation. DESIGN, SETTING, AND PARTICIPANTS: We computed the mutation rate in different sized uveal melanomas using previously published large data sets. Tumor volume was estimated using the spherical cap method. We also calculated the metastatic rate using an updated data set of patients with uveal melanoma with known mutations in BAP1, SF3B1, and EIF1AX provided by the Rotterdam Ocular Melanoma Study Group. Data were analyzed from 2 studies, one taking place from August 25, 1970, to August 27, 2008, and the other taking place between 1993 and 2013. Data were analyzed between 2016 and 2017. MAIN OUTCOMES AND MEASURES: Mutation rates and metastic rates. RESULTS: Based on the 5-year metastatic rates, mutation rates ranged from 1.09 × 10−8 to 7.86 × 10−7 per cell division, using our calculation algorithm. A higher mutation rate was found for tumors with smaller thicknesses. EIF1AX mutations were not exclusive of other mutations because 2 cases with EIF1AX mutations and metastasis also had BAP1 mutations. None of the tumors with only an EIF1AX mutation metastasized. After plotting the yearly metastatic rate vs time after treatment, we observed a small peak at 1 year and a large peak at 3.5 years after treatment for BAP1 mutations, with peaks between 2 and 3 years and at 7 years for SF3B1 mutations. CONCLUSIONS AND RELEVANCE: We observed a higher mutation rate for smaller tumors, which may be explained by a greater number of cell divisions occurring during the expansion phase of smaller uveal melanomas. Regarding time to clinically detected metastases, the first 2 peaks appear to be associated with BAP1-mutated tumors and the late peak to SF3B1-mutated tumors.
Approximately 25% of all uveal melanoma (UM) contain driver mutations in the gene encoding the spliceosome factor
, and whilst patients with such
mutations generally have an intermediate risk on ...developing metastatic disease, a third of these patients develop early metastasis within 5 years after diagnosis. We therefore investigated whether clinical and/or genetic variables could be indicative of short progression-free survival (PFS < 60 months) or long PFS (PFS ≥ 60 months) for
-mutated (
) UM patients. We collected 146
UM from our Rotterdam Ocular Melanoma Studygroup (ROMS) database and external published datasets. After stratification of all
UM using short PFS vs. long PFS, only largest tumor diameter (LTD) was significantly larger (mean: 17.7 mm (±2.8 SD) in the short PFS
group vs. the long PFS group (mean: 14.7 (±3.7 SD,
= 0.001). Combined ROMS and The Cancer Genome Atlas (TCGA) transcriptomic data were evaluated, and we identified
-specific canonical transcripts (e.g., a low expression of
indicative for early-onset metastatic disease) or distinct expression of
UM aberrant transcripts, indicative of early- or late-onset or no metastatic
UM.
Uveal melanoma (UM) is characterized by multiple chromosomal rearrangements and recurrent mutated genes. The aim of this study was to investigate if copy number variations (CNV) alone and in ...combination with other genetic and clinico-histopathological variables can be used to stratify for disease-free survival (DFS) in enucleated patients with UM.
We analyzed single nucleotide polymorphisms (SNP) array data of primary tumors and other clinical variables of 214 UM patients from the Rotterdam Ocular Melanoma Study (ROMS) cohort. Nonweighted hierarchical clustering of SNP array data was used to identify molecular subclasses with distinct CNV patterns. The subclasses associate with mutational status of BAP1, SF3B1, or EIF1AX. Cox proportional hazard models were then used to study the predictive performance of SNP array cluster-, mutation-, and clinico-histopathological data, and their combination for study endpoint risk.
Five clusters with distinct CNV patterns and concomitant mutations in BAP1, SF3B1, or EIF1AX were identified. The sample's cluster allocation contributed significantly to mutational status of samples in predicting the incidence of metastasis during a median of 45.6 (interquartile range IQR: 24.7-81.8) months of follow-up (P < 0.05) and vice versa. Furthermore, incorporating all data sources in one model yielded a 0.797 C-score during 100 months of follow-up.
UM has distinct CNV patterns that correspond to different mutated driver genes. Incorporating clinico-histopathological, cluster and mutation data in the analysis results in good performance for UM-related DFS prediction.
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