Germline mutation in the TP53 gene in uveal melanoma

We performed comprehensive molecular analysis of five cases of metastasizing uveal malignant melanoma (UM) (fresh-frozen samples) with an NGS panel of 73 genes. A likely pathogenic germline TP53 mutation c.760A > G (p.I254V) was found in two tumor samples and matched nontumor tissue. In three cases, pathogenic BAP1 mutation was detected together with germline missense variants of uncertain significance in ATM. All cases carried recurrent activating GNAQ or GNA11 mutation. Moreover, we analyzed samples from another 16 patients with primary UM by direct Sanger sequencing focusing only on TP53 coding region. No other germline TP53 mutation was detected in these samples. Germline TP53 mutation, usually associated with Li-Fraumeni syndrome, is a rare event in UM. To the best of our knowledge, only one family with germline TP53 mutation has previously been described. In our study, we detected TP53 mutation in two patients without known family relationship. The identification of germline aberrations in TP53 or BAP1 is important to identify patients with Li-Fraumeni syndrome or BAP1 cancer syndrome, which is also crucial for proper genetic counseling.

Patients with germline variants in TP53. In two of five patients with mUMs, identical germline TP53 mutation NM_001126112.2: c.760A > G, p.I254V was detected (representative visualization in IGV and confirmation by Sanger sequencing is in Supplementary Fig. S1). These two patients are unrelated according to family history, and the probability of kinship was furthermore excluded by a bioinformatics approach. One of the carriers was a male patient with primary diagnosis of uveal melanoma at the age of 54, his father had a brain tumor at 60 and his mother had breast cancer at 54. Both parents were not genetically tested and archive tissue for retrospective mutation analysis of TP53 is unavailable. The second carrier was a female patient diagnosed with uveal melanoma at the age of 39, a family history of cancer has not been demonstrated. Immunohistochemical (IHC) analysis performed on liver tissue slides showed wild-type expression of the p53 protein.
To screen germline TP53 mutations in a larger cohort, the TP53 mutation analysis by direct Sanger sequencing was performed in additional 16 nontumor tissue from unrelated patients with UM. No other patient with TP53 germline mutation was found.
Both mUM samples with germline TP53 mutation did not have a deletion of chromosome 3 and carried a duplication of 8q (Supplementary Fig. S2 and Supplementary Table S2). One of them also carried a somatic mutation in SF3B1 (NM_012433.2: c.1874G > T, p.R625L). Further, one of these patients carried a germline mutation in MSH2 (NM_000251.2: c.4G > A, p.A2T). This variant is probably damaging according to the in silico prediction programs, but IHC examination showed intact nuclear expression of all MMR proteins (MSH2, MSH6, MLH1, PMS2) and fragment analysis showed microsatellite stable phenotype.
Patients with somatic mutations in BAP1 and germline variants in ATM. A germline mutation of ATM and a somatic mutation in BAP1 were found in three patients. None of these patients have a mutation of TP53. One patient carried a mutation in ATM (NM_000051.3:c.5975A > C, p.K1992T) and had a somatic frameshift mutation in BAP1 (NM_004656.2:c.79delG, p.V27Cfs*45, VAF 29.21%). Other patient had a somatic mutation in the BAP1 first coding amino acid that leads to the change of methionine to lysine (c.2T > A, p.?, VAF 60.78%). CNV analyses suggested a partial deletion of chromosome 3, and the patient carried a germline missense mutation in ATM (c.5558A > T, p.D1853V) and a germline tandem duplication of 56 bp in exon 48 of the ATM gene (c.7010_7065dup56, p.?) ( Supplementary Fig. S3). Exon 48 of the ATM gene is a part of the regulatory FAT domain that inhibits ATM kinase activity until the occurrence of DNA damage 13 . This patient developed uveal melanoma relatively late at age 66, and breast cancer was diagnosed at 70.

Discussion
The Cancer Genome Atlas (TCGA) has recently published comprehensive data of 80 uveal melanomas and suggested four prognostic subtypes 11 . The most prevalent somatic mutations were GNAQ (guanine nucleotide-binding protein G(q) subunit α) (50%), GNA11 (guanine nucleotide-binding protein subunit α-11) (45%), BAP1 (BRCA1 associated protein-1 (ubiquitin carboxy-terminal hydrolase) (32.5%), SF3B1 (splicing factor 3b, subunit 1) (22.5%), and EIF1AX (Eukaryotic Translation Initiation Factor 1 A, X-Linked) (12.5%). Mutant GNAQ was shown to activate the MAPK pathway and it may also have important effects on other pathways such as the phosphatidylinositol-calcium second messenger system 2 . Hot-spot mutations affecting amino acid Glutamine at codon 209 in GNAQ or its paralog GNA11 are mutually exclusive, they have also been detected in benign uveal nevi and are not sufficient for full malignant transformation to melanoma 2,10 . Total loss of function of BAP1, that codes the protein involved in DNA damage control, correlates with increased metastatic potential. Partial or complete monosomy of chromosome 3, where BAP1 (3p21.31-p21.2) is located, is a relatively common event in metastasizing uveal melanoma. Other common chromosomal changes include gain or loss of 1p, 6q, 8q, 8p and less frequently 9p, and 16q. There is a myriad of combinations of such cytogenetic changes. Monosomy of chromosome 3, gain of 8q, epithelioid-mixed cell type and/or larger tumor diameter were strongly associated with a poor prognosis and potential to metastasize 2,14,15 .
In our study, comprehensive molecular analysis of five cases of mUMs was performed. We detected germline TP53 mutation in 2/5 patients and germline ATM mutation together with somatic BAP1 alteration in another 3/5 patients. Somatic GNAQ/GNA11 recurrent mutation was detected in all 5 cases. Further, somatic or germline variants were detected in BARD1, CDH1, MET, MMR genes, PARD3, SF3B1, SNAI3 (Table 1). Based on the unexpected finding of germline TP53 mutation, we analyzed another 16 patients with UM focusing only on germline TP53 mutations. However, no other TP53 mutation was found.
The TP53 gene is highly polymorphic in coding and noncoding regions and some of these polymorphisms have been shown to increase cancer susceptibility 16 . The frequency of de novo TP53 germline mutation has been estimated up to 30%, which is very high compared with the frequency of mutations in other tumor suppressor genes 17,18 . Germline mutations in TP53 are linked to Li-Fraumeni syndrome (LFS). Germline missense mutations are the most common TP53 variants, occurring in approximately 70% of cases and mainly altering residues within the DNA-binding domain 19 . Patients with LFS are predisposed to a wide variety of cancer types, with early onset, and with the potential for multiple primary cancer sites, including breast cancer, brain tumor, soft tissues cancer, adrenocortical carcinoma and other types 16 . Association of cutaneous malignant melanoma with LFS is relatively rare [20][21][22] . Moreover, a case of mucosal melanoma has been associated with LFS 23 .
Somatic mutations in the TP53 gene are one of the most frequent alterations in human cancers. Somatic TP53 mutations were detected in 12-19% cases of cutaneous malignant melanoma [24][25][26] and also described in melanocytic tumor originating in the central nervous system 27 . In uveal melanoma, the occurrence of a somatic mutation is rare [28][29][30][31][32][33] . However, most researchers use immunostaining with an antibody against p53 protein to detect aberrant protein expression in UM instead of mutation analysis by sequencing of the TP53 gene. According to the dataset from IARC TP53 database, detected missense mutation show positive IHC results in 88% of cases. Not only TP53 mutations but also a disturbed p53 pathway can result in abnormal p53 expression 34 .
On the contrary, germline TP53 mutations in UM is, according to the literature, exceedingly rare and has been described only once in a British family with four generations burdened with uveal melanoma 35  Czech family with LFS (family without evidence of uveal melanoma) and causal TP53 mutation p.I254V 36 . The result of functional analysis of p53 transactivation ability in yeast (FASAY) 37 has confirmed that p.I254V is a fully inactivating mutation. Codon 254 is buried in DNA binding domain and located in conserved beta-sheet structure according to the 3D model of p53 (Supplementary Fig. S4) 38 . Any change in this conserved domain could potentially lead to change of conformation, even though amino acids Isoleucine and Valine has similar physicochemical properties. On the contrary, in silico analyses suggested deleterious effect of this variant due to its impact on protein structure and function. The ClinVar database (https://www.ncbi.nlm.nih.gov/clinvar/, accessed November 2017) contains data from two submitters and indicates uncertain significance of this germline TP53 variant. Halvorsen et al. detected germline variant p.I254V in TP53 in 3 out of 394 patients with lung cancer and designated this variant as polymorphism. Nevertheless, we need to take into consideration that this opinion is not based on strong arguments 39 . On the other hand, in the same codon 254 another two missense variants p.I254T and p.I254L were described, either somatic or germline in LFS family 40 , that were considered pathogenic throughout the databases and the literature. Somatic mutation p.I254V has been described according to the IARC TP53 database (http://p53.iarc.fr; assessed December 2017) in seven different tumors so far ( Table 2).
Inherited pathogenic ATM mutation is the cause of autosomal recessive disease ataxia-telangiectasia which predisposes the individuals for increased cancer risk 41,42 . Germline or somatic mutations in ATM have not been described in uveal melanoma so far. In our study, we detected germline ATM variants in three patients with metastatic uveal melanoma. Nevertheless, all variants were of unknown significance or likely benign. Three of them have been described previously and their population mutant allele frequency (MAF) is low: p.K1992T, MAF(gnomAD) = 0.0003; p.D1853V, MAF(gnomAD) = 0.004915, and p.L508F, which is only described in database FLOSSIES MAF = 0.0001. In all three of our cases, ATM mutations cooccurred with pathogenic somatic variants in the BAP1 gene and two tumors have partial loss of chromosome 3, which are also relatively common event in metastasizing uveal melanoma, both associated with worse prognosis. Not only somatic but also germline mutation of BAP1 gene may occur in UM and the majority of familial UM with germline BAP1 mutation is associated with BAP1-tumor predisposition syndrome, which also increases the risk of the atypical Spitz tumors, malignant mesothelioma, and cutaneous melanoma 43 .

Conclusion
In conclusion, the results of our study have shown that genetic changes occurring in UM can be very heterogeneous. In three tumors and/or metastases we detected pathogenic somatic mutations in BAP1, which is not an unexpected finding, but these mutations occur in all three patients together with the germline missense variant in ATM. Despite the fact that detected ATM variants are of uncertain significance, this finding deserves further research because ATM mutations have never been reported in UM to date. Moreover, we have found germline TP53 mutation in two patients. Germline TP53 aberration has been described in UM in only one family so far, but the true incidence of TP53 mutations in UM is difficult to estimate due to the sparse studies focusing on this topic. However, the identification of germline TP53 or BAP1 mutations is important to be able to identify patients with Li-Fraumeni syndrome or BAP1 cancer syndrome and is the first step for proper genetic counseling and management of the patients and family members. We are well aware of the limitations of our study, which are mainly due to the small sample set. Nevertheless, we believe that the results of our study broaden the knowledge of molecular changes occurring in such a rare tumor as UM.

Material and Methods
Patients and samples. Archive files of the Bank of Biological Material of the First Faculty of Medicine, Charles University, Prague, were searched for uveal malignant melanomas or their metastasis. Five fresh frozen liver metastasis (stored in liquid nitrogen at −180 °C) of uveal melanoma (mUM) from 5 patients were found. For all 5 mUM cases, formalin-fixed paraffin-embedded tissue (FFPE) blocks of liver metastasis were also available for subsequent IHC analysis. Corresponding fresh-frozen nontumor tissue was available in 4/5 patients and genomic DNA from blood was available in 1/5 patient. Characteristics of the patients and tumors are summarized in Table 1   Biostatistical analysis. NextGENe ® Software was used for the analysis of the sequencing data, and CNV analysis. The Pindel tool was used to detect break points of large deletions and medium sized insertions 44 . The R package SNPRelate was performed to exclude unknown family relationships 45 . Copy number variation. Used custom NGS panel was not primarily designed for analysis of copy number variation (CNV), so only regions with sufficient coverage by target genes were analyzed. Each region of our custom panel and their chromosomal location are listed in Supplementary Table S6. CNV analysis was performed focused on chromosome 3, 6, 8, 16, according to the current knowledge about cytogenetic changes in uveal melanoma. CNV tool Dispersion and Hidden Markov Model (HMM; part of NextGENe Software) was used to evaluate CNV variations between tumor tissue and corresponding nontumor tissue (mUM_1, mUM_2, mUM_3, mUM_4) and in case mUM_5 for comparison were used normalized coverage of the pool of other 4 nontumor samples.
In silico prediction tools. In order to assess the impact of detected missense variants, we employed several widely used in silico prediction programs or databases, which are imported in NextGENe Software.
Mutant allele frequencies for ATM aberrations were searched in databases The Genome Aggregation Database (gnomAD) and Fabulous Ladies Over Seventy database (FLOSSIES).