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Analysis of uveal melanomas and paired constitutional DNA for exclusion of a BAP1-tumor predisposition syndrome

Analysis of uveal melanomas and paired constitutional DNA for exclusion of a BAP1-tumor... Uveal melanoma (UM) is a rare tumor originating from melanocytic cells in the eye. Familial aggregation of UM is rare and can occur as part of the tumor predisposition syndrome BAP1-TPDS. However, family history alone will only identify a subset of patients with BAP1-TPDS. In the present study, we used sequential testing of tumor and blood DNA from UM patients for differential diagnosis of BAP1-TPDS. The study group was an unselected prospective cohort of patients from whom UM tissue was available. First, chromosome 3 status in tumor DNA was determined in all 140 patients who consented to participate. As tumors with disomy 3 rarely show BAP1 alterations, sequence analysis of this gene was performed in the 72 tumors with monosomy 3 (M3) or partial M3 only. We identified oncogenic BAP1 alterations in 52 of these tumors (72%). Targeted sequencing of DNA from matched peripheral blood showed pathogenic variants in two patients (3.8%) thus proving BAP1-TPDS. Only one of these two patients also had a medical history suggestive of this syndrome. Conversely, in three patients known to have had additional tumors before diagnosis of UM, constitutional heterozygosity for a BAP1 mutation was excluded. Altogether, in 50 patients we could exclude BAP1-TPDS with high diagnostic certainty. The results of our study support that genetic testing for BAP1-TPDS should be offered to all patients with UM. Moreover, as genetic information from the tumor can help exclude heritable risk, the strategy for analysis should include efforts to obtain tumor samples for testing. Keywords Uveal melanoma · BAP1 gene · BAP1-TPDS · Predictive testing Introduction to tan [3]. These phenotypic features are heritable traits, and in fact, some polymorphic genetic variants linked to pig- Uveal melanoma (UM) is a malignant tumor that originates mentation are associated with a risk to UM [4]. However, from melanocytic cells in the iris, ciliary body, and choroid the strength of these associations is low, with odds ratios of the eye [1]. UM is a rare tumor [1]—and, consequently, typically less than 2 [4]. Consequently, at the level of the average individual risk is low. However, the incidence of individual, the effect of these polymorphic genetic variants UM shows marked differences worldwide with 1.3–8.6 cases on risk to UM has no clinical consequences. per million per year in Europe and 0.2–0.3 cases per million The rare observations of families showing aggregation in Africa and Asia [2]. This variation appears to be associ- of patients with UM have suggested that heritable predis- ated with differences in phenotypic features related to mel- position to UM can be transmitted as an autosomal domi- anocyte functioning such as iris color, skin color, and ability nant trait albeit with incomplete penetrance, i.e., absence of phenotype in some carriers [5]. The prototypical example of autosomal dominant tumor predisposition is heritable * Dietmar R. Lohmann retinoblastoma [6]. The hypothesis that the development of Dietmar.Lohmann@uni-due.de this tumor depends on a two-step mutation process [7] has guided strategies that led to the identification of the RB1 Institute of Human Genetics, University Hospital Essen, University Duisburg-Essen, Hufelandstr. 55, 45147 Essen, gene that is responsible for a heritable predisposition to ret- Germany inoblastoma. Using a similar approach, the BAP1 was identi- Department of Ophthalmology, University Hospital Essen, fied as a likely candidate gene for a heritable predisposition University Duisburg-Essen, Hufelandstr. 55, 45147 Essen, to UM [8, 9]. The deubiquitinase BAP1 is multifunctional Germany Vol.:(0123456789) 1 3 Y. A. Abbassi et al. protein with tumor suppressor activity involved in chromatin material was available to participate in the study. From these, remodeling, DNA damage response, cell cycle regulation, 140 patients agreed. Tumor samples were obtained either by cell death, and differentiation [10, 11]. It turned out that biopsy sampling, by enucleation or endoresection, depend- genetic variants of BAP1 can cause high risk not only to UM ing on treatment of the primary tumor. Chromosome 3 status but also to a spectrum of other neoplasia [8, 9, 12, 13]. It (disomy 3, or monosomy 3) of all tumor samples was ana- is well established that heritable risk to UM can be part of lyzed by microsatellite analysis (MSA). BAP1 sequencing a tumor predisposition syndrome with variable manifesta- was performed on DNA from all samples with monosomy 3 tion and incomplete penetrance (BAP1-tumor predisposition (M3) or partial losses of chromosome 3 (partM3) (n = 72). syndrome, BAP1-TPDS), and the number of families with known oncogenic variants of the BAP1 gene has risen fast Samples and genetic analysis [14]. Families suspected to have BAP1-TPDS can benefit from Genomic DNA was isolated from enucleated tumors by the genetic testing. Specifically, if diagnostic testing shows that phenol–chloroform method as described previously [18]. a patient with UM has a BAP1-TPDS, this justifies repeated DNA from biopsy samples was isolated using the QIAamp surveillance examinations for early detection of metachro- DNA Mini Kit (Qiagen, Hilden, Germany). DNA from blood nous tumors, although details of the monitoring strategy was isolated using the FlexiGene kit (Qiagen). The chromo- need to be developed [15]. Identification of the family- some 3 status of all tumor samples was determined by STR- specific pathogenic BAP1 gene variant facilitates predic- genotyping (microsatellite analysis, MSA) using eight chro- tive testing in relatives. In cancer predisposition syndromes mosome 3 markers as described elsewhere [19]. To detect with incomplete penetrance, relatives heterozygous for tumors with isodisomy 3 all tumors showing loss of het- the mutation have an increased risk [16]. With predictive erozygosity (LOH) of chromosome 3 markers by MSA were genetic testing, it is possible to exclude an increased risk in subjected to chromosome 3 MLPA (MRC Holland, probe- relatives. In addition to the benefit of psychological relief, mix P027-C2). BAP1 mutation screening was performed by overall health care cost is likely to be lower than without Sanger sequencing in DNA from 72 UM with M3 or partM3 genetic testing [16]. as described in Martin et al., [20]. In samples without clear Only a small proportion of patients with UM have a fam- results by Sanger sequencing, NGS panel sequencing was ily history suggestive of BAP1-TPDS, i.e., most patients performed. Copy number analysis of all BAP1 exons was have isolated UM. Genetic testing has shown that some determined by Multiplex ligation-dependent probe ampli- of these patients are heterozygous for BAP1 variants that fication (MLPA) using SALSA MLPA probemix P417-B2 cause BAP1-TPDS [8, 9] and this finding establishes the BAP1 (MRC Holland, Amsterdam, The Netherlands) fol- diagnosis of BAP1-TPDS. However, if the genetic analysis lowing the manufacturer's instructions. is performed on DNA from blood only and fails to identify a pathogenic BAP1 variant, then a BAP1-TPDS is less likely Panel sequencing but not excluded. Because the spectrum of BAP1 variants XT HS associated with BAP1-TPDS is very heterogeneous, it has to In this study, Agilent SureSelect target enrichment sys- be expected that the false-negative rate of mutation screen- tem was used to enrich the genomic regions of interest. The ing tests is not negligible. In patients with isolated retino- customized panel was designed to capture all coding regions blastoma, this problem is solved by mutation identification of BAP1, SF3B1, EIF1AX, CYSLTR2, PCLB4, RB1, and on DNA from the tumor first, followed by targeted testing exons 4 and 5 of the GNAQ and GNA11 genes. About 100 ng in DNA from blood [17]. With this diagnostic strategy, it genomic tumor DNA was subjected to library preparation is possible to exclude an inherited predisposition in most according to the manufacturer's instructions (protocol ver- patients with isolated unilateral retinoblastoma. The goal sion A1, July 2017). Briefly, genomic DNA was fragmented of the study presented here was to apply this strategy for using a Covaris S220 to an average size of 150 to 200 bp, differential diagnosis of BAP1-TPDS in patients with uveal and the fragments were ligated to molecular barcodes. The melanoma. captured fragments enriched for the target sequences were sequenced on Illumina MiSeq by paired-end sequencing with 2 × 150 bp. We obtained a coverage of > 400 × for all Material and methods coding regions in each sample. For mapping of the reads and mutation calling, the fastq files were analyzed using SureCall Patients (version 4.1.1.5, Agilent). Sanger sequencing was used to re- sequence regions showing mutations by Panel Sequencing We invited all UM patients treated in our clinic between in tumor DNA and to sequence the respective blood DNA October 2014 and October 2016 and from whom tumor of the patient. The pathogenicity of the germline variants 1 3 Analysis of uveal melanomas and paired constitutional DNA for exclusion of a BAP1‑tumor… was determined using InterVar (https:// winte r v ar . w glab. or g/) Chromosome 3 status which is based on the ACMG/AMP 2015 guidelines [21]. For evaluating the oncogenicity of somatic variants we have Mutational inactivation of the BAP1 gene is rarely seen adhered to the applicable criteria of the ACMG guidelines: in the class of UMs with disomy 3 (D3) if isodisomy 3 is Nonsense, frameshift, exon skipping and deletion variants in excluded [22, 23]. To focus the analytical efforts on sam- conjunction with LOH of BAP1 are classified as oncogenic ples likely to have BAP1 gene alterations we determined mutations. Missense variations in BAP1 with a ClinVar or the chromosome 3 status in tumors before further mutation COSMIC entry are classified as oncogenic if in addition analysis. Genotype data of polymorphic STR-loci from computational data indicate impaired protein function. 134 patients met our technical quality criteria for deter- mining the chromosome 3 status. These criteria require that a minimum number of loci are heterozygous in DNA Results from blood. Uveal melanomas from 62 patients (46%) showed no loss of heterozygosity at any informative locus Participants and, therefore, were classified as disomy 3 tumors (Fig.  1). In tumors from 61 patients (46%), monosomy 3 was diag- All patients (n = 265) who requested prognostic biomarker nosed because of loss of heterozygosity at all informa- testing (chromosome 3 status in tumor DNA) were eligi- tive loci (Fig. 1). In UMs from the remaining 11 patients ble (148 males (55.8%), median age = 61.3  years, inter- (6%), only some of the informative loci on chromosome quartile range (IQR) = 15.8 years and 117 female, median 3 showed allele loss (Fig. 1). This pattern of allele loss is age = 61.7 years, IQR = 21.2 years). Of these, 140 (52.8%) typical for uveal melanomas with deletions only of parts consented to participate in this study (83 males (59.3%) with of chromosome 3 (partM3). median age at diagnosis = 60.8 years and IQR = 14 years; 57 females with median age at diagnosis = 57.9 years and IQR = 20.1 years). 140 patients with Uveal Melanoma (UM) 62 Patients 61 Patients 11 Patients 6 Patients Tumors with Tumors with Tumors with partial tumor analysis failed Disomy 3 Monosomy 3 chr. 3 allele loss 49 Patients 3 Patients 12 Patients 8 Patients no BAP1 Variant BAP1 Variant BAP1 Variant no BAP1 Variant detected in Tumor detected in Tumor detected in Tumor detected in Tumor 2 Patients 2 Patients BAP1- 50 Patients BAP1- BAP1 Variant of TPDS excluded Fig. 1 Overview of the study cohort and the grouping of patients depending on the chromosome 3 status and the mutation status of the primary tumor 1 3 Y. A. Abbassi et al. two patients, we could not track the origin of the variants Mutation analysis of the BAP1 gene in uveal melanoma any further. BAP1 mutation analysis was performed in UM with mono- Functional types and localization of oncogenic somy 3 or partial deletions of chromosome 3 (72 tumors). alterations Sanger sequencing was chosen as the first-line method of sequencing analysis. Those samples that did not meet the Missense and in-frame length variants (11 and 5, respec- quality criteria for mutation analysis, which require, among tively) were identified in samples from 16 patients, and all other parameters, that sequence data has been obtained for were of somatic origin (Table 1). Amino acids predicted to all coding and invariant splice site regions of the BAP1 gene, be altered by these mutations were all located in the N-ter- were subjected to panel sequencing which included the minal UCH domain of the BAP1 protein (Fig. 2a). One mis- BAP1 gene. Copy number variant detection by MLPA was sense variant, c.188C > G [p.(Ser63Cys)], was identified performed on samples that did not show a oncogenic small in tumors of two patients and is also listed as a recurrent variant. Alterations of the BAP1 gene were identified in 52 somatic variant (n = 5) in the Catalogue Of Somatic Muta- of the 72 tumors with M3 or partM3 (72%). In the tumors tions In Cancer (COSMIC V90_38_MUTANTCENSUS). with partM3 the percentage of samples with BAP1 mutation Of the other 14 variants predicted to alter only single or was lower (27%) than in the M3 group. few amino acids of the BAP1 gene 9 variants (56%) are also In 50 of 52 tumors we detected oncogenic BAP1 altera- listed in COSMIC. The interpretation of one missense muta- tions (Table  1). Alterations identified in these 50 tumors tion p.(Ser98Arg) although located in the UCH domain and included two deletions that spanned one or more exons. The present on the background of chromosome 3 loss remains remaining 48 alterations were small variants. In these tumors uncertain as computational data do not indicate a severe the signal of the normal allele was low compared to that of impairment of protein function. the variant allele. This result is in line with hemizygosity at Tumors from 32 patients had variants resulting in pre- the BAP1 locus, which is expected in tumors with mono- mature termination codons (3 nonsense, 19 frame-shift, somy 3. In tumors with alterations only of parts of chromo- and ten splice site variants; Table 1). The locations of these some 3 this result suggests that the BAP1 locus is within terminations showed no clustering to particular domains of regions of allele loss. the BAP1 protein (Fig. 2b). No premature termination was Two of 52 tumors did not show a bona fide pathogenic located within the last exon and the 50 bp 3′-parts of the alteration but an identical single base substitution at the -4 penultimate exon of the BAP1, which are regions expected position of the splice donor site of exon 17. This position is not to elicit nonsense-mediated decay [26]. One of the vari- not part of the invariant splice donor sequence motive and ants resulting in premature termination codons, c.588G > A the base substitution does not create an alternative donor [p.(Trp196Ter)], was identified in tumors of two patients and site. In silico analysis of this intronic variant have previously is also listed as a recurrent somatic variant (n = 4) in COS- shown that it is not expected to affect splicing [24]. In both MIC. However, in all, only 4 of the 28 (14%) premature ter- patients, this variant allele was also present in the constitu- mination variants identified here are also listed in COSMIC. tional DNA and, therefore, it is plausible that this alteration One tumor (TP1) showed a deletion of the splice accep- is a rare and likely neutral polymorphic variant. tor-site of intron 5 and parts of exon 5 (Table  1). The expected consequence of this variant is a loss of exon 5 Analysis of DNA from blood for patient‑specific from the mRNA and, on translation, an in-frame deletion of variant BAP1 alleles Leu86 (CTG) to Glu125(GAG) (Fig. 2c). These amino acids are part of the N-terminal UCH domain of the BAP1 protein. We performed BAP1 mutation analysis targeting the onco- genic variants detected in the tumor on DNA from matched peripheral blood (50 patients). In 48 patients (96%) results Relations between constitutional BAP1 genotype and phenotypic features showed that the oncogenic variant identified in the tumor resulted from a somatic mutation in the patient. Two patients The ages at diagnosis of the two patients with pathogenic (4%) showed heterozygosity for the oncogenic variant identi- fied in the tumor, thus indicating a germ-line origin (TP7, BAP1 germ-line variants ranked at positions 4 and 21 within the group of 50 patients with BAP1 variants. Their age rank TP24). Both variants were classie fi d as pathogenic by “Inter - Var” based on ACMG criteria. One of the germline muta- positions within the whole group of 140 patients were simi- lar (positions 12 and 71 of 140). In the density plots of age tions p.(Ser126Glnfs*16) has been identified in a patient with mesothelioma, previously [25]. As we had no access to at diagnosis, the two patients with germ-line variants also do not appear to be outliers (Fig. 3). constitutional DNA from parents or other relatives of these 1 3 Analysis of uveal melanomas and paired constitutional DNA for exclusion of a BAP1‑tumor… 1 3 Table 1 BAP1 sequence variants in 52 uveal melanomas ID Chr.3 BAP1 variant HGVS_p Functional description origin Seq. method Mutation ID ClinVar ClinVar Mutation ID cosmic COSMIC status recur- recurrences rences TP1 M3 exon 5 deletion p.(Leu86_Glu125del) In-frame-deletion Somatic Panel NA – NA – TP2 M3 exon 8–10 deletion p.(Gly194_Asp311) In-frame-deletion Somatic Sanger NA – NA – TP3 M3 c.1984-19_1984-4del p.(Ile662Alafs*5) Exon-skipping fs Somatic Sanger NA – NA – TP4 M3 c.784-2A > G p.(Leu262Metfs*22) Exon-skipping fs Somatic Sanger NA – NA – TP5 PartM3pq c.122 + 3_122 + 11del p.(Gly23Alafs*23) Exon-skipping fs Somatic Sanger NA – NA – TP6 M3 c.1729G > C p.(Tyr418Glyfs*64) Exon-skipping fs Somatic Sanger 114,540,411 1 COSV56239857 1 TP7 M3 c.1984-2A > C p.(Ile662Alafs*5) Exon-skipping fs Germ line Sanger NA – NA – TP8 M3 c.67 + 13_67 + 53del p.(Gly13Alafs*29) Exon-skipping fs Somatic Sanger NA – NA – TP9 M3 c.376-5_376-2del p.(Ser126Alafs*6) Exon-skipping fs Somatic Sanger NA – NA – TP10 M3 c.540_580 + 20delinsC p.(Pro147Alafs*46) Exon-skipping fs Somatic Panel NA – NA – TP11 M3 c.363_375 + 41delTTT p.(Leu86_Glu125del) In-frame-deletion Somatic Panel NA – NA – TP12 M3 c.256-12_310del p.(Leu86_Glu125del) Exon-skipping in-frame Somatic Sanger NA – NA – TP13 M3 c.1049_1050delinsT p.(Pro350Leufs*12) Frameshift Somatic Sanger NA – NA – TP14 M3 c.39_42del p.(Leu14Serfs*57) Frameshift Somatic Sanger NA – NA – TP15 M3 c.500_511del p.(Ser10_Gly13delfs*67) Frameshift Somatic Sanger NA – NA – TP16 PartM3p c.1654dup p.(Asp552Glyfs*15) Frameshift Somatic Sanger NA – NA – TP17 M3 c.577del p.(His193Metfs*38) Frameshift Somatic Sanger NA – COSV105168035 1 TP18 M3 c.740del p.(Val247Glyfs*2) Frameshift Somatic Sanger NA – NA – TP19 M3 c.1366del p.(Gln456Argfs*115) Frameshift Somatic Sanger NA – NA – TP20 M3 c.400del p.(Ala134Profs*53) Frameshift Somatic Sanger NA – NA – TP21 PartM3q c.585del p.(Trp196Glyfs*35) Frameshift Somatic Sanger NA – NA – TP22 M3 c.843_846delinsTA p.(Pro282Argfs*24) Frameshift Somatic Sanger NA – NA – TP23 M3 c.26_47del p.(Glu9Alafs*56) Frameshift Somatic Sanger NA – NA – TP24 M3 c.376_377del p.(Ser126Glnfs*16) Frameshift Germ line Sanger NA – NA – TP25 M3 c.350_375 + 45del p.(Asp117_Glu126fs*142) Frameshift Somatic Sanger NA – NA – TP26 M3 c.1965_1966del p.(Lys656Glufs*7) Frameshift Somatic Sanger 114,534,267 1 NA – TP27 M3 c.170_179del p.(Arg57Glnfs*12) Frameshift Somatic Sanger NA – NA – TP28 M3 c.880_899del p.(Leu294Glyfs*6) Frameshift Somatic Sanger NA – NA – TP29 M3 c.570_580del p.(Ile191Alafs*48) Frameshift Somatic Sanger NA – NA – TP30 M3 c.1762_1796delinsA p.(Pro588Argfs*18) Frameshift Somatic Sanger NA – NA – TP31 M3 c.1636del p.(Tyr546Thrfs*25) Frameshift Somatic Sanger NA – NA – TP32 M3 c.522_530del p.(Pro175_Thr177)del In-frame Somatic Sanger NA – NA – TP33 M3 c.182_200delinsC p.(Lys61_Asp67delinsThr) In-frame Somatic Sanger NA – NA – TP34 M3 c.520_525del p.(Val174_Pro175del) In-frame Somatic Sanger NA – NA – TP35 M3 c.692_706del p.(Met231_delinsAsp236 In-frame Somatic Sanger NA – NA – Asn) Y. A. Abbassi et al. 1 3 Table 1 (continued) ID Chr.3 BAP1 variant HGVS_p Functional description origin Seq. method Mutation ID ClinVar ClinVar Mutation ID cosmic COSMIC status recur- recurrences rences TP36 M3 c.486_507del p.(Thr164_Phe170del) In-frame Somatic Sanger NA – NA – TP37 M3 c.535C > T p.(Arg179Trp) Missense Somatic Sanger NA – COSV56230719 4 TP38 M3 c.299 T > G p.(Leu100Arg) Missense Somatic Panel NA – COSV56235221 1 TP39 M3 c.188C > G p.(Ser63Cys) Missense Somatic Sanger 114,533,374 5 COSV56230632 1 TP40 M3 c.505C > T p.(His169Tyr) Missense Somatic Sanger 114,535,404 5 COSV56233385 5 TP41 M3 c.518A > C p.(Tyr173Ser) Missense Somatic Sanger 114,538,920 2 COSV56238100 2 TP42 M3 c.509 T > C (p.Phe170Ser) Missense Somatic Sanger NA – NA no TP43 M3 c.92A > G p.(Glu31Gly) Missense Somatic Sanger 114,541,608 1 COSV56241651 2 TP44 M3 c.422A > G p.(His141Arg) Missense Somatic Sanger 114,532,971 3 COSV56229904 3 TP45 M3 c.188C > G p.(Ser63Cys) Missense Somatic Sanger 114,533,374 5 COSV56230632 1 TP46 M3 c.272G > A p.(Cys91Tyr) Missense Somatic Sanger 114,541,032 2 COSV56240775 2 TP47 M3 c.284C > G p.(Ser98Arg) Missense Somatic Sanger NA – NA – TP48 M3 c.588G > A p.(Trp196*) Nonsense Somatic Sanger 114,534,223 4 COSV56231737 4 TP49 M3 c.588G > A p.(Trp196*) Nonsense Somatic Sanger 114,534,223 4 COSV56231737 4 TP50 M3 c.1447C > T p.(Gln483*) Nonsense Somatic Sanger 114,536,409 1 NA – TP51 M3 c.2057-4G > T exon 17 splice acceptor No effect Germ line Panel NA – NA – TP52 M3 c.2057-4G > T exon 17 splice acceptor No effect Germ line Sanger NA – NA – Mutation description refers to BAP1 transcript reference sequence NM_004656.3. In both tumors with partial loss of 3p regions, BAP1 gene is included in deleted region. With the exception of the variant indentified in TP7 all missense mutations are classified as oncogenic according to ACMG criteria as applicable Analysis of uveal melanomas and paired constitutional DNA for exclusion of a BAP1‑tumor… Fig. 2 Map of BAP1 oncogenic sequence variants in 50 uveal melanomas. a Black triangles: location of somatic missense or in-frame variants. b and c Black circles: location of premature stop codons due to nonsense, frameshift or splice variants caused by somatic altera- tions. Red circles: location of premature stop codons due to nonsense, frameshift or splice variants caused by constitu- tional alterations. Grey bar: in-frame loss of codons due to splice variant caused by somatic alteration. in-frame 1 729 BAP1 protein N-terminal UCH domain Key residues (Q82, C91, H169, D184) UCH37-like domain Coiled coil domain HCF1-binding domain NLS-motifs Fig. 3 Density plots of age at diagnosis of 140 patients included in the study grouped by chromosome 3 status Disomy 3 Monosomy 3Partial allele loss At the time of diagnosis of the primary tumor, data on cutaneous malignant melanoma. In the two other patients medical history concerning tumors outside of the eye was with diagnosis of cutaneous malignant melanoma prior to available from 100 patients. Two patients had a history of uveal melanoma, a common pathogenic BAP1 gene germline renal cell cancer diagnosed prior to uveal melanoma but only variant as root cause is highly unlikely because the BAP1 one of these two patients was heterozygous for a pathogenic gene mutations that we identified in their tumors were not BAP1 germ-line mutation. This patient also had a history of present in DNA from blood and, therefore, of somatic origin. 1 3 Age at diagnosis/years Y. A. Abbassi et al. However, as we cannot exclude low-dose mosaicism and In the tumors with partM3 the percentage of samples with thus it is conceivable that tumors can originate from cells of BAP1 mutation was much lower (27%) compared to M3 a mutant sector that share a oncogenic BAP1 gene somatic tumors which is in concordance with the observation that variant. Data on family history was incomplete for most most of these tumors show a mutation pattern typical for the patients. Of note, the father of a BAP1 heterozygous patient UM with D3 [20]. Between reported studies the proportion in our study had died from pleural mesothelioma, a tumor of tumors with M3 with a oncogenic BAP1 alteration varies type that is very rare in the general population but relatively and the detection rate in our study is well within this range frequent in families with BAP1-TPDS (for review see [14]). [23, 28]. It is conceivable that some tumors with M3 may have BAP1 gene alterations that are not within the scope of Discussion the detection methods used here. Specifically, we expect an improved rate of detection if sequencing libraries cover the In this study we used genetic testing of tumors for molecular complete BAP1 gene region instead of focusing enrichment differential diagnosis of BAP1-TPDS in patients with UM. of the exons only. The need for extension of the scope of We adapted a stepwise testing strategy similar to the one that analysis by NGS has already been pointed out by Field et al. is used for routine in genetic testing in retinoblastoma to the 2018 [23, 28]. Another advantage of NGS-based analysis is requirements specific for uveal melanoma. The initial analy - a lower detection limit compared to Sanger sequencing. This sis step identifies tumors with complete or partial monosomy allows the detection of sequence variants in heterogeneous 3 and, in the next step, DNA from these tumors is analyzed tumor tissue containing high proportion of normal cells [23] for BAP1 gene alterations. With decreasing sequencing costs thus increasing the proportion of fully informative cases, due to NGS, we would suggest sequencing of tumor and although it does not lead to the discovery of additional her- blood tissue simultaneously instead of one after another itable cases. which is more time efficient. A BAP1-TPDS is excluded In 2 patients (4% of 50 patients with BAP1 mutant tumors, in patients in whom a somatic origin of these alterations 1.4% of all 140 patients) we determined heterozygosity for is established by analysis of constitutional DNA. With this pathogenic BAP1 gene variants and this established the diag- three-step strategy, we could safely exclude a BAP1-TPDS nosis of a BAP1-TPDS. Both variants are expected to result in 50 of 72 (70%) patients with a M3 or partM3 UM. Since in premature termination which is the predominant type of germ-line variant alleles of the BAP1 gene are rare in UM consequence of pathogenic BAP1 alterations. Premature ter- patients with D3 tumors (odds ratio 0.12, derived from data mination was also the most frequent biological consequence published by Ewens 2018 [22]), TPDS is also unlikely in the among the BAP1 germ-line variants reported by Ewens et al. group of 62 patients with D3 tumors. However, it remains [22]. By contrast, studies restricted to mutation analysis on to be shown if extending the diagnostic strategy to patients DNA from blood tend to report a higher proportion of mis- with UM with D3 leads to detection of further BAP1-TPDS sense type variants and these include a high proportion of with a frequency higher than those in the normal (UM-free) variants of uncertain pathogenic significance (VUS) [22]. population [22]. In fact, 608 of 738 (82%) gene variants in the BAP1 coding Most of the alterations with confirmed somatic origin region listed in ClinVar in the context of heritable tumor resulted in premature termination outside of the regions susceptibility are VUS (https://www .ncbi. nlm. nih. go v/clin v that are expected to evade nonsense mediated mRNA decay ar, query of 14.7.2021). Using our strategy, which requires [26] and, therefore, are expected to result in loss of func- initial sequencing of tumor DNA, only half as much VUS tion, which is typical for the spectrum of BAP1 alterations are expected due to loss of one BAP1 allele in most tumors. in cancer. All amino acids altered by missense substitution In other words, results of mutation analysis on DNA from or short in-frame losses belonged to the ubiquitin C-terminal blood only are often insufficient to provide the evidence hydrolase (UCH) domain of the BAP1 protein. This catalytic needed to demonstrate or exclude a BAP1-TPDS with high domain plays a key role by mediating deubiquitination of confidence. In vitro assays that assess if some functions of chromatin elements such as H2A [27]. Most of the missense BAP1 protein are altered depending on certain alterations substitutions identified here have been reported as somatic can provide additional data in support of the pathogenicity mutations in other tumors and this also supports that these of exonic BAP1 variants [29, 30]. However, we agree that alterations can be classified as pathogenic. Recurrence of “further research into BAP1 functions is needed” to arrive somatic missense mutations was also found in a recent report at “a consensus on mandatory experimental assays to assess by Ewens et al. [22]. Thus, it appears that the set of onco- VUS” [30]. genic missense mutations of the BAP1 gene is smaller than Only one of the two patients that we identified to be the set of variants that result in premature termination. heterozygous for a pathogenic BAP1 alteration was also We did not identify oncogenic BAP1 alterations in DNA positive for features proposed as referral guidelines for from 14 to 8 tumors (29%) with M3 or partM3, respectively. genetic diagnostics of a BAP1-TPDS [31]. This patient 1 3 Analysis of uveal melanomas and paired constitutional DNA for exclusion of a BAP1‑tumor… Consent for publication All patients included in this study signed was diagnosed with CMM and RCC (at age 28 and informed consent regarding publishing their data. 39 years, respectively) prior to the diagnosis of UM (at age 59 years). Moreover, one parent of this patient had Open Access This article is licensed under a Creative Commons Attri- died of pleural mesothelioma, a rare cancer that is frequent bution 4.0 International License, which permits use, sharing, adapta- in patients with a BAP1-TPDS. The medical and family tion, distribution and reproduction in any medium or format, as long history of the other BAP1-TPDS patient identified in our as you give appropriate credit to the original author(s) and the source, study did not meet the referral criteria proposed by Chau provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are et al. [31]. However, with a relatively young age at diag- included in the article's Creative Commons licence, unless indicated nosis of UM (40 years) this patient qualifies according to otherwise in a credit line to the material. If material is not included in more relaxed guidelines [9]. From recent studies it appears the article's Creative Commons licence and your intended use is not that population-based analyses, which avoid the ascertain- permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a ment biases introduced by selection of high-risk patients, copy of this licence, visit http://cr eativ ecommons. or g/licen ses/ b y/4.0/ . are required to obtain a better understanding of the BAP1- TPDS [22]. In other cancer predisposition syndromes such as hereditable breast and ovarian cancer (HBOC) it References has turned out that initial estimates of penetrance, which were derived from observations of familial aggregation of 1. Kaliki S, Shields CL (2017) Uveal melanoma: relatively rare cancer cases, were inflated. Unbiased data are required to but deadly cancer. Eye (Lond) 31(2):241–257. https:// doi. org/ model the cancer risk of individuals who are heterozygous 10. 1038/ eye. 2016. 275 2. Krantz BA, Dave N, Komatsubara KM, Marr BP, Carvajal RD for a pathogenic BAP1 allele. Such a statistical model is (2017) Uveal melanoma: epidemiology, etiology, and treatment essential to derive surveillance plans for early detection. of primary disease. Clin Ophthalmol 11:279–289. https:// doi. The results of our study support that genetic diagnosis of org/ 10. 2147/ OPTH. S89591 a BAP1-TPDS should be offered to all patients with UM. 3. Weis E, Shah CP, Lajous M, Shields JA, Shields CL (2006) The association between host susceptibility factors and uveal Moreover, the testing strategy should include efforts to melanoma: a meta-analysis. Arch Ophthalmol 124(1):54–60. obtain genetic information from the tumor. https:// doi. org/ 10. 1001/ archo pht. 124.1. 54 4. Ferguson R, Vogelsang M, Ucisik-Akkaya E et al (2016) Genetic Acknowledgements We thank Prof. Dr. Hebebrandt for providing the markers of pigmentation are novel risk loci for uveal melanoma. nucleus for the initiation of the study. Sci Rep 6:31191. https:// doi. org/ 10. 1038/ srep3 1191 5. Lynch HT, Krush AJ (1968) Heredity and malignant mela- Author contributions Material preparation, data collection, and analy- noma: implications for early cancer detection. Can Med Assoc sis were performed by all authors. The first draft of the manuscript was J 99(1):17–21 written by Dietmar Lohmann and all authors commented on previous 6. Vogel F (1954) Genetics and mutation rate of retinoblastoma versions of the manuscript. All authors read and approved the final (glioma retinae), with general remarks on methods of determin- manuscript. DL: conceptualization; MZ, YAA: methodology; DL: for- ing mutation rate in humans. Z Mensch Vererb Konstitutionsl mal analysis and investigation; DL: writing—original draft preparation; 32(4):308–336 MZ: writing—review and editing; DL: funding acquisition. 7. Knudson AG Jr (1971) Mutation and cancer: statistical study of retinoblastoma. Proc Natl Acad Sci U S A 68(4):820–823. https:// doi. org/ 10. 1073/ pnas. 68.4. 820 Funding Open Access funding enabled and organized by Projekt 8. Wiesner T, Obenauf AC, Murali R et al (2011) Germline muta- DEAL. This work was supported by Deutsche Krebshilfe (Grant tions in BAP1 predispose to melanocytic tumors. Nat Genet No.70110960). 43(10):1018–1021. https:// doi. org/ 10. 1038/ ng. 910 9. Abdel-Rahman MH, Pilarski R, Cebulla CM et  al (2011) Data availability Not applicable. Germline BAP1 mutation predisposes to uveal melanoma, lung adenocarcinoma, meningioma, and other cancers. J Code availability Not applicable. Med Genet 48(12):856–859. https:// doi. or g/ 10. 1136/ jmedg enet- 2011- 100156 10. Han A, Purwin TJ, Aplin AE (2021) Roles of the BAP1 tumor Declarations suppressor in cell metabolism. Cancer Res 81(11):2807–2814. https:// doi. org/ 10. 1158/ 0008- 5472. CAN- 20- 3430 Conflicts of interest The authors have no relevant financial or non- 11. Carbone M, Harbour JW, Brugarolas J et al (2020) Biologi- financial interests to disclose. cal mechanisms and clinical significance of BAP1 mutations in human cancer. Cancer Discov 10(8):1103–1120. https://d oi.o rg/ Ethical approval This study was performed in line with the principles 10. 1158/ 2159- 8290. CD- 19- 1220 of the Declaration of Helsinki. Approval was granted by the Ethics 12. Carbone M, Ferris LK, Baumann F et al (2012) BAP1 cancer Committee of the University Hospital Essen (13–5462-B0). syndrome: malignant mesothelioma, uveal and cutaneous mela- noma, and MBAITs. J Transl Med 10:179. https:// doi. org/ 10. Consent to participate Written informed consent to participate was 1186/ 1479- 5876- 10- 179 given by every patient included in this study. 1 3 Y. A. Abbassi et al. 13. Testa JR, Cheung M, Pei J et  al (2011) Germline BAP1 melanoma. Nat Commun 9(1):116. https:// doi. or g/ 10. 1038/ mutations predispose to malignant mesothelioma. Nat Genet s41467- 017- 02428-w 43(10):1022–1025. https:// doi. org/ 10. 1038/ ng. 912 24. Betti M, Casalone E, Ferrante D et al (2015) Inference on ger- 14. Walpole S, Pritchard AL, Cebulla CM et al (2018) Comprehen- mline BAP1 mutations and asbestos exposure from the analysis sive study of the clinical phenotype of germline BAP1 variant- of familial and sporadic mesothelioma in a high-risk area. Genes carrying families worldwide. J Natl Cancer Inst 110(12):1328– Chromosomes Cancer 54(1):51–62. https:// doi. org/ 10. 1002/ gcc. 1341. https:// doi. org/ 10. 1093/ jnci/ djy171 22218 15. Star P, Goodwin A, Kapoor R et al (2018) Germline BAP1- 25. Carbone M, Pass HI, Ak G et al (2022) Medical and surgical positive patients: the dilemmas of cancer surveillance and a care of patients with mesothelioma and their relatives carrying proposed interdisciplinary consensus monitoring strategy. Eur germline BAP1 mutations. J Thorac Oncol 17(7):873–889. https:// J Cancer 92:48–53. https:// doi. org/ 10. 1016/j. ejca. 2017. 12. 022doi. org/ 10. 1016/j. jtho. 2022. 03. 014 16. Noorani HZ, Khan HN, Gallie BL, Detsky AS (1996) Cost com- 26. Lindeboom RG, Supek F, Lehner B (2016) The rules and impact parison of molecular versus conventional screening of relatives of nonsense-mediated mRNA decay in human cancers. Nat Genet at risk for retinoblastoma. Am J Hum Genet 59(2):301–307 48(10):1112–1118. https:// doi. org/ 10. 1038/ ng. 3664 17. Lohmann DR, Gerick M, Brandt B et al (1997) Constitutional 27. Okino Y, Machida Y, Frankland-Searby S, Machida YJ (2015) RB1-gene mutations in patients with isolated unilateral retino- BRCA1-associated protein 1 (BAP1) deubiquitinase antagonizes blastoma. Am J Hum Genet 61(2):282–294. https:// doi. org/ 10. the ubiquitin-mediated activation of FoxK2 target genes. J Biol 1086/ 514845 Chem 290(3):1580–1591. https:// doi. or g/ 10. 1074/ jbc. M114. 18. Tschentscher F, Husing J, Holter T et al (2003) Tumor clas- 609834 sification based on gene expression profiling shows that uveal 28. van de Nes JA, Nelles J, Kreis S et al (2016) Comparing the prog- melanomas with and without monosomy 3 represent two distinct nostic value of BAP1 mutation pattern, chromosome 3 status, and entities. Cancer Res 63(10):2578–2584 BAP1 immunohistochemistry in uveal melanoma. Am J Surg 19. Tschentscher F, Prescher G, Zeschnigk M, Horsthemke B, Lohm- Pathol 40(6):796–805. https:// doi. org/ 10. 1097/ PAS. 00000 00000 ann DR (2000) Identification of chromosomes 3, 6, and 8 aberra- 000645 tions in uveal melanoma by microsatellite analysis in comparison 29. Hong JH, Chong ST, Lee PH et al (2020) Functional characterisa- to comparative genomic hybridization. Cancer Genet Cytogenet tion guides classification of novel BAP1 germline variants. NPJ 122(1):13–17. https:// doi. org/ 10. 1016/ s0165- 4608(00) 00266-1 Genom Med 5:50. https:// doi. org/ 10. 1038/ s41525- 020- 00157-6 20. Martin M, Masshofer L, Temming P et al (2013) Exome sequenc- 30. Repo P, Jarvinen RS, Jantti JE et  al (2019) Population-based ing identifies recurrent somatic mutations in EIF1AX and SF3B1 analysis of BAP1 germline variations in patients with uveal mel- in uveal melanoma with disomy 3. Nat Genet 45(8):933–936. anoma. Hum Mol Genet 28(14):2415–2426. https:// doi. org/ 10. https:// doi. org/ 10. 1038/ ng. 26741093/ hmg/ ddz076 21. Richards S, Aziz N, Bale S et al (2015) Standards and guidelines 31. Chau C, van Doorn R, van Poppelen NM et al (2019) Families for the interpretation of sequence variants: a joint consensus rec- with BAP1-tumor predisposition syndrome in The Netherlands: ommendation of the American College of Medical Genetics and path to identification and a proposal for genetic screening guide- Genomics and the Association for Molecular Pathology. Genet lines. Cancers (Basel). https:// doi. org/ 10. 3390/ cance rs110 81114 Med 17(5):405–424. https:// doi. org/ 10. 1038/ gim. 2015. 30 22. Ewens KG, Lalonde E, Richards-Yutz J, Shields CL, Ganguly Publisher's Note Springer Nature remains neutral with regard to A (2018) Comparison of germline versus somatic BAP1 muta- jurisdictional claims in published maps and institutional affiliations. tions for risk of metastasis in uveal melanoma. BMC Cancer 18(1):1172. https:// doi. org/ 10. 1186/ s12885- 018- 5079-x 23. Field MG, Durante MA, Anbunathan H et  al (2018) Punc- tuated evolution of canonical genomic aberrations in uveal 1 3 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Familial Cancer Springer Journals

Analysis of uveal melanomas and paired constitutional DNA for exclusion of a BAP1-tumor predisposition syndrome

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Abstract

Uveal melanoma (UM) is a rare tumor originating from melanocytic cells in the eye. Familial aggregation of UM is rare and can occur as part of the tumor predisposition syndrome BAP1-TPDS. However, family history alone will only identify a subset of patients with BAP1-TPDS. In the present study, we used sequential testing of tumor and blood DNA from UM patients for differential diagnosis of BAP1-TPDS. The study group was an unselected prospective cohort of patients from whom UM tissue was available. First, chromosome 3 status in tumor DNA was determined in all 140 patients who consented to participate. As tumors with disomy 3 rarely show BAP1 alterations, sequence analysis of this gene was performed in the 72 tumors with monosomy 3 (M3) or partial M3 only. We identified oncogenic BAP1 alterations in 52 of these tumors (72%). Targeted sequencing of DNA from matched peripheral blood showed pathogenic variants in two patients (3.8%) thus proving BAP1-TPDS. Only one of these two patients also had a medical history suggestive of this syndrome. Conversely, in three patients known to have had additional tumors before diagnosis of UM, constitutional heterozygosity for a BAP1 mutation was excluded. Altogether, in 50 patients we could exclude BAP1-TPDS with high diagnostic certainty. The results of our study support that genetic testing for BAP1-TPDS should be offered to all patients with UM. Moreover, as genetic information from the tumor can help exclude heritable risk, the strategy for analysis should include efforts to obtain tumor samples for testing. Keywords Uveal melanoma · BAP1 gene · BAP1-TPDS · Predictive testing Introduction to tan [3]. These phenotypic features are heritable traits, and in fact, some polymorphic genetic variants linked to pig- Uveal melanoma (UM) is a malignant tumor that originates mentation are associated with a risk to UM [4]. However, from melanocytic cells in the iris, ciliary body, and choroid the strength of these associations is low, with odds ratios of the eye [1]. UM is a rare tumor [1]—and, consequently, typically less than 2 [4]. Consequently, at the level of the average individual risk is low. However, the incidence of individual, the effect of these polymorphic genetic variants UM shows marked differences worldwide with 1.3–8.6 cases on risk to UM has no clinical consequences. per million per year in Europe and 0.2–0.3 cases per million The rare observations of families showing aggregation in Africa and Asia [2]. This variation appears to be associ- of patients with UM have suggested that heritable predis- ated with differences in phenotypic features related to mel- position to UM can be transmitted as an autosomal domi- anocyte functioning such as iris color, skin color, and ability nant trait albeit with incomplete penetrance, i.e., absence of phenotype in some carriers [5]. The prototypical example of autosomal dominant tumor predisposition is heritable * Dietmar R. Lohmann retinoblastoma [6]. The hypothesis that the development of Dietmar.Lohmann@uni-due.de this tumor depends on a two-step mutation process [7] has guided strategies that led to the identification of the RB1 Institute of Human Genetics, University Hospital Essen, University Duisburg-Essen, Hufelandstr. 55, 45147 Essen, gene that is responsible for a heritable predisposition to ret- Germany inoblastoma. Using a similar approach, the BAP1 was identi- Department of Ophthalmology, University Hospital Essen, fied as a likely candidate gene for a heritable predisposition University Duisburg-Essen, Hufelandstr. 55, 45147 Essen, to UM [8, 9]. The deubiquitinase BAP1 is multifunctional Germany Vol.:(0123456789) 1 3 Y. A. Abbassi et al. protein with tumor suppressor activity involved in chromatin material was available to participate in the study. From these, remodeling, DNA damage response, cell cycle regulation, 140 patients agreed. Tumor samples were obtained either by cell death, and differentiation [10, 11]. It turned out that biopsy sampling, by enucleation or endoresection, depend- genetic variants of BAP1 can cause high risk not only to UM ing on treatment of the primary tumor. Chromosome 3 status but also to a spectrum of other neoplasia [8, 9, 12, 13]. It (disomy 3, or monosomy 3) of all tumor samples was ana- is well established that heritable risk to UM can be part of lyzed by microsatellite analysis (MSA). BAP1 sequencing a tumor predisposition syndrome with variable manifesta- was performed on DNA from all samples with monosomy 3 tion and incomplete penetrance (BAP1-tumor predisposition (M3) or partial losses of chromosome 3 (partM3) (n = 72). syndrome, BAP1-TPDS), and the number of families with known oncogenic variants of the BAP1 gene has risen fast Samples and genetic analysis [14]. Families suspected to have BAP1-TPDS can benefit from Genomic DNA was isolated from enucleated tumors by the genetic testing. Specifically, if diagnostic testing shows that phenol–chloroform method as described previously [18]. a patient with UM has a BAP1-TPDS, this justifies repeated DNA from biopsy samples was isolated using the QIAamp surveillance examinations for early detection of metachro- DNA Mini Kit (Qiagen, Hilden, Germany). DNA from blood nous tumors, although details of the monitoring strategy was isolated using the FlexiGene kit (Qiagen). The chromo- need to be developed [15]. Identification of the family- some 3 status of all tumor samples was determined by STR- specific pathogenic BAP1 gene variant facilitates predic- genotyping (microsatellite analysis, MSA) using eight chro- tive testing in relatives. In cancer predisposition syndromes mosome 3 markers as described elsewhere [19]. To detect with incomplete penetrance, relatives heterozygous for tumors with isodisomy 3 all tumors showing loss of het- the mutation have an increased risk [16]. With predictive erozygosity (LOH) of chromosome 3 markers by MSA were genetic testing, it is possible to exclude an increased risk in subjected to chromosome 3 MLPA (MRC Holland, probe- relatives. In addition to the benefit of psychological relief, mix P027-C2). BAP1 mutation screening was performed by overall health care cost is likely to be lower than without Sanger sequencing in DNA from 72 UM with M3 or partM3 genetic testing [16]. as described in Martin et al., [20]. In samples without clear Only a small proportion of patients with UM have a fam- results by Sanger sequencing, NGS panel sequencing was ily history suggestive of BAP1-TPDS, i.e., most patients performed. Copy number analysis of all BAP1 exons was have isolated UM. Genetic testing has shown that some determined by Multiplex ligation-dependent probe ampli- of these patients are heterozygous for BAP1 variants that fication (MLPA) using SALSA MLPA probemix P417-B2 cause BAP1-TPDS [8, 9] and this finding establishes the BAP1 (MRC Holland, Amsterdam, The Netherlands) fol- diagnosis of BAP1-TPDS. However, if the genetic analysis lowing the manufacturer's instructions. is performed on DNA from blood only and fails to identify a pathogenic BAP1 variant, then a BAP1-TPDS is less likely Panel sequencing but not excluded. Because the spectrum of BAP1 variants XT HS associated with BAP1-TPDS is very heterogeneous, it has to In this study, Agilent SureSelect target enrichment sys- be expected that the false-negative rate of mutation screen- tem was used to enrich the genomic regions of interest. The ing tests is not negligible. In patients with isolated retino- customized panel was designed to capture all coding regions blastoma, this problem is solved by mutation identification of BAP1, SF3B1, EIF1AX, CYSLTR2, PCLB4, RB1, and on DNA from the tumor first, followed by targeted testing exons 4 and 5 of the GNAQ and GNA11 genes. About 100 ng in DNA from blood [17]. With this diagnostic strategy, it genomic tumor DNA was subjected to library preparation is possible to exclude an inherited predisposition in most according to the manufacturer's instructions (protocol ver- patients with isolated unilateral retinoblastoma. The goal sion A1, July 2017). Briefly, genomic DNA was fragmented of the study presented here was to apply this strategy for using a Covaris S220 to an average size of 150 to 200 bp, differential diagnosis of BAP1-TPDS in patients with uveal and the fragments were ligated to molecular barcodes. The melanoma. captured fragments enriched for the target sequences were sequenced on Illumina MiSeq by paired-end sequencing with 2 × 150 bp. We obtained a coverage of > 400 × for all Material and methods coding regions in each sample. For mapping of the reads and mutation calling, the fastq files were analyzed using SureCall Patients (version 4.1.1.5, Agilent). Sanger sequencing was used to re- sequence regions showing mutations by Panel Sequencing We invited all UM patients treated in our clinic between in tumor DNA and to sequence the respective blood DNA October 2014 and October 2016 and from whom tumor of the patient. The pathogenicity of the germline variants 1 3 Analysis of uveal melanomas and paired constitutional DNA for exclusion of a BAP1‑tumor… was determined using InterVar (https:// winte r v ar . w glab. or g/) Chromosome 3 status which is based on the ACMG/AMP 2015 guidelines [21]. For evaluating the oncogenicity of somatic variants we have Mutational inactivation of the BAP1 gene is rarely seen adhered to the applicable criteria of the ACMG guidelines: in the class of UMs with disomy 3 (D3) if isodisomy 3 is Nonsense, frameshift, exon skipping and deletion variants in excluded [22, 23]. To focus the analytical efforts on sam- conjunction with LOH of BAP1 are classified as oncogenic ples likely to have BAP1 gene alterations we determined mutations. Missense variations in BAP1 with a ClinVar or the chromosome 3 status in tumors before further mutation COSMIC entry are classified as oncogenic if in addition analysis. Genotype data of polymorphic STR-loci from computational data indicate impaired protein function. 134 patients met our technical quality criteria for deter- mining the chromosome 3 status. These criteria require that a minimum number of loci are heterozygous in DNA Results from blood. Uveal melanomas from 62 patients (46%) showed no loss of heterozygosity at any informative locus Participants and, therefore, were classified as disomy 3 tumors (Fig.  1). In tumors from 61 patients (46%), monosomy 3 was diag- All patients (n = 265) who requested prognostic biomarker nosed because of loss of heterozygosity at all informa- testing (chromosome 3 status in tumor DNA) were eligi- tive loci (Fig. 1). In UMs from the remaining 11 patients ble (148 males (55.8%), median age = 61.3  years, inter- (6%), only some of the informative loci on chromosome quartile range (IQR) = 15.8 years and 117 female, median 3 showed allele loss (Fig. 1). This pattern of allele loss is age = 61.7 years, IQR = 21.2 years). Of these, 140 (52.8%) typical for uveal melanomas with deletions only of parts consented to participate in this study (83 males (59.3%) with of chromosome 3 (partM3). median age at diagnosis = 60.8 years and IQR = 14 years; 57 females with median age at diagnosis = 57.9 years and IQR = 20.1 years). 140 patients with Uveal Melanoma (UM) 62 Patients 61 Patients 11 Patients 6 Patients Tumors with Tumors with Tumors with partial tumor analysis failed Disomy 3 Monosomy 3 chr. 3 allele loss 49 Patients 3 Patients 12 Patients 8 Patients no BAP1 Variant BAP1 Variant BAP1 Variant no BAP1 Variant detected in Tumor detected in Tumor detected in Tumor detected in Tumor 2 Patients 2 Patients BAP1- 50 Patients BAP1- BAP1 Variant of TPDS excluded Fig. 1 Overview of the study cohort and the grouping of patients depending on the chromosome 3 status and the mutation status of the primary tumor 1 3 Y. A. Abbassi et al. two patients, we could not track the origin of the variants Mutation analysis of the BAP1 gene in uveal melanoma any further. BAP1 mutation analysis was performed in UM with mono- Functional types and localization of oncogenic somy 3 or partial deletions of chromosome 3 (72 tumors). alterations Sanger sequencing was chosen as the first-line method of sequencing analysis. Those samples that did not meet the Missense and in-frame length variants (11 and 5, respec- quality criteria for mutation analysis, which require, among tively) were identified in samples from 16 patients, and all other parameters, that sequence data has been obtained for were of somatic origin (Table 1). Amino acids predicted to all coding and invariant splice site regions of the BAP1 gene, be altered by these mutations were all located in the N-ter- were subjected to panel sequencing which included the minal UCH domain of the BAP1 protein (Fig. 2a). One mis- BAP1 gene. Copy number variant detection by MLPA was sense variant, c.188C > G [p.(Ser63Cys)], was identified performed on samples that did not show a oncogenic small in tumors of two patients and is also listed as a recurrent variant. Alterations of the BAP1 gene were identified in 52 somatic variant (n = 5) in the Catalogue Of Somatic Muta- of the 72 tumors with M3 or partM3 (72%). In the tumors tions In Cancer (COSMIC V90_38_MUTANTCENSUS). with partM3 the percentage of samples with BAP1 mutation Of the other 14 variants predicted to alter only single or was lower (27%) than in the M3 group. few amino acids of the BAP1 gene 9 variants (56%) are also In 50 of 52 tumors we detected oncogenic BAP1 altera- listed in COSMIC. The interpretation of one missense muta- tions (Table  1). Alterations identified in these 50 tumors tion p.(Ser98Arg) although located in the UCH domain and included two deletions that spanned one or more exons. The present on the background of chromosome 3 loss remains remaining 48 alterations were small variants. In these tumors uncertain as computational data do not indicate a severe the signal of the normal allele was low compared to that of impairment of protein function. the variant allele. This result is in line with hemizygosity at Tumors from 32 patients had variants resulting in pre- the BAP1 locus, which is expected in tumors with mono- mature termination codons (3 nonsense, 19 frame-shift, somy 3. In tumors with alterations only of parts of chromo- and ten splice site variants; Table 1). The locations of these some 3 this result suggests that the BAP1 locus is within terminations showed no clustering to particular domains of regions of allele loss. the BAP1 protein (Fig. 2b). No premature termination was Two of 52 tumors did not show a bona fide pathogenic located within the last exon and the 50 bp 3′-parts of the alteration but an identical single base substitution at the -4 penultimate exon of the BAP1, which are regions expected position of the splice donor site of exon 17. This position is not to elicit nonsense-mediated decay [26]. One of the vari- not part of the invariant splice donor sequence motive and ants resulting in premature termination codons, c.588G > A the base substitution does not create an alternative donor [p.(Trp196Ter)], was identified in tumors of two patients and site. In silico analysis of this intronic variant have previously is also listed as a recurrent somatic variant (n = 4) in COS- shown that it is not expected to affect splicing [24]. In both MIC. However, in all, only 4 of the 28 (14%) premature ter- patients, this variant allele was also present in the constitu- mination variants identified here are also listed in COSMIC. tional DNA and, therefore, it is plausible that this alteration One tumor (TP1) showed a deletion of the splice accep- is a rare and likely neutral polymorphic variant. tor-site of intron 5 and parts of exon 5 (Table  1). The expected consequence of this variant is a loss of exon 5 Analysis of DNA from blood for patient‑specific from the mRNA and, on translation, an in-frame deletion of variant BAP1 alleles Leu86 (CTG) to Glu125(GAG) (Fig. 2c). These amino acids are part of the N-terminal UCH domain of the BAP1 protein. We performed BAP1 mutation analysis targeting the onco- genic variants detected in the tumor on DNA from matched peripheral blood (50 patients). In 48 patients (96%) results Relations between constitutional BAP1 genotype and phenotypic features showed that the oncogenic variant identified in the tumor resulted from a somatic mutation in the patient. Two patients The ages at diagnosis of the two patients with pathogenic (4%) showed heterozygosity for the oncogenic variant identi- fied in the tumor, thus indicating a germ-line origin (TP7, BAP1 germ-line variants ranked at positions 4 and 21 within the group of 50 patients with BAP1 variants. Their age rank TP24). Both variants were classie fi d as pathogenic by “Inter - Var” based on ACMG criteria. One of the germline muta- positions within the whole group of 140 patients were simi- lar (positions 12 and 71 of 140). In the density plots of age tions p.(Ser126Glnfs*16) has been identified in a patient with mesothelioma, previously [25]. As we had no access to at diagnosis, the two patients with germ-line variants also do not appear to be outliers (Fig. 3). constitutional DNA from parents or other relatives of these 1 3 Analysis of uveal melanomas and paired constitutional DNA for exclusion of a BAP1‑tumor… 1 3 Table 1 BAP1 sequence variants in 52 uveal melanomas ID Chr.3 BAP1 variant HGVS_p Functional description origin Seq. method Mutation ID ClinVar ClinVar Mutation ID cosmic COSMIC status recur- recurrences rences TP1 M3 exon 5 deletion p.(Leu86_Glu125del) In-frame-deletion Somatic Panel NA – NA – TP2 M3 exon 8–10 deletion p.(Gly194_Asp311) In-frame-deletion Somatic Sanger NA – NA – TP3 M3 c.1984-19_1984-4del p.(Ile662Alafs*5) Exon-skipping fs Somatic Sanger NA – NA – TP4 M3 c.784-2A > G p.(Leu262Metfs*22) Exon-skipping fs Somatic Sanger NA – NA – TP5 PartM3pq c.122 + 3_122 + 11del p.(Gly23Alafs*23) Exon-skipping fs Somatic Sanger NA – NA – TP6 M3 c.1729G > C p.(Tyr418Glyfs*64) Exon-skipping fs Somatic Sanger 114,540,411 1 COSV56239857 1 TP7 M3 c.1984-2A > C p.(Ile662Alafs*5) Exon-skipping fs Germ line Sanger NA – NA – TP8 M3 c.67 + 13_67 + 53del p.(Gly13Alafs*29) Exon-skipping fs Somatic Sanger NA – NA – TP9 M3 c.376-5_376-2del p.(Ser126Alafs*6) Exon-skipping fs Somatic Sanger NA – NA – TP10 M3 c.540_580 + 20delinsC p.(Pro147Alafs*46) Exon-skipping fs Somatic Panel NA – NA – TP11 M3 c.363_375 + 41delTTT p.(Leu86_Glu125del) In-frame-deletion Somatic Panel NA – NA – TP12 M3 c.256-12_310del p.(Leu86_Glu125del) Exon-skipping in-frame Somatic Sanger NA – NA – TP13 M3 c.1049_1050delinsT p.(Pro350Leufs*12) Frameshift Somatic Sanger NA – NA – TP14 M3 c.39_42del p.(Leu14Serfs*57) Frameshift Somatic Sanger NA – NA – TP15 M3 c.500_511del p.(Ser10_Gly13delfs*67) Frameshift Somatic Sanger NA – NA – TP16 PartM3p c.1654dup p.(Asp552Glyfs*15) Frameshift Somatic Sanger NA – NA – TP17 M3 c.577del p.(His193Metfs*38) Frameshift Somatic Sanger NA – COSV105168035 1 TP18 M3 c.740del p.(Val247Glyfs*2) Frameshift Somatic Sanger NA – NA – TP19 M3 c.1366del p.(Gln456Argfs*115) Frameshift Somatic Sanger NA – NA – TP20 M3 c.400del p.(Ala134Profs*53) Frameshift Somatic Sanger NA – NA – TP21 PartM3q c.585del p.(Trp196Glyfs*35) Frameshift Somatic Sanger NA – NA – TP22 M3 c.843_846delinsTA p.(Pro282Argfs*24) Frameshift Somatic Sanger NA – NA – TP23 M3 c.26_47del p.(Glu9Alafs*56) Frameshift Somatic Sanger NA – NA – TP24 M3 c.376_377del p.(Ser126Glnfs*16) Frameshift Germ line Sanger NA – NA – TP25 M3 c.350_375 + 45del p.(Asp117_Glu126fs*142) Frameshift Somatic Sanger NA – NA – TP26 M3 c.1965_1966del p.(Lys656Glufs*7) Frameshift Somatic Sanger 114,534,267 1 NA – TP27 M3 c.170_179del p.(Arg57Glnfs*12) Frameshift Somatic Sanger NA – NA – TP28 M3 c.880_899del p.(Leu294Glyfs*6) Frameshift Somatic Sanger NA – NA – TP29 M3 c.570_580del p.(Ile191Alafs*48) Frameshift Somatic Sanger NA – NA – TP30 M3 c.1762_1796delinsA p.(Pro588Argfs*18) Frameshift Somatic Sanger NA – NA – TP31 M3 c.1636del p.(Tyr546Thrfs*25) Frameshift Somatic Sanger NA – NA – TP32 M3 c.522_530del p.(Pro175_Thr177)del In-frame Somatic Sanger NA – NA – TP33 M3 c.182_200delinsC p.(Lys61_Asp67delinsThr) In-frame Somatic Sanger NA – NA – TP34 M3 c.520_525del p.(Val174_Pro175del) In-frame Somatic Sanger NA – NA – TP35 M3 c.692_706del p.(Met231_delinsAsp236 In-frame Somatic Sanger NA – NA – Asn) Y. A. Abbassi et al. 1 3 Table 1 (continued) ID Chr.3 BAP1 variant HGVS_p Functional description origin Seq. method Mutation ID ClinVar ClinVar Mutation ID cosmic COSMIC status recur- recurrences rences TP36 M3 c.486_507del p.(Thr164_Phe170del) In-frame Somatic Sanger NA – NA – TP37 M3 c.535C > T p.(Arg179Trp) Missense Somatic Sanger NA – COSV56230719 4 TP38 M3 c.299 T > G p.(Leu100Arg) Missense Somatic Panel NA – COSV56235221 1 TP39 M3 c.188C > G p.(Ser63Cys) Missense Somatic Sanger 114,533,374 5 COSV56230632 1 TP40 M3 c.505C > T p.(His169Tyr) Missense Somatic Sanger 114,535,404 5 COSV56233385 5 TP41 M3 c.518A > C p.(Tyr173Ser) Missense Somatic Sanger 114,538,920 2 COSV56238100 2 TP42 M3 c.509 T > C (p.Phe170Ser) Missense Somatic Sanger NA – NA no TP43 M3 c.92A > G p.(Glu31Gly) Missense Somatic Sanger 114,541,608 1 COSV56241651 2 TP44 M3 c.422A > G p.(His141Arg) Missense Somatic Sanger 114,532,971 3 COSV56229904 3 TP45 M3 c.188C > G p.(Ser63Cys) Missense Somatic Sanger 114,533,374 5 COSV56230632 1 TP46 M3 c.272G > A p.(Cys91Tyr) Missense Somatic Sanger 114,541,032 2 COSV56240775 2 TP47 M3 c.284C > G p.(Ser98Arg) Missense Somatic Sanger NA – NA – TP48 M3 c.588G > A p.(Trp196*) Nonsense Somatic Sanger 114,534,223 4 COSV56231737 4 TP49 M3 c.588G > A p.(Trp196*) Nonsense Somatic Sanger 114,534,223 4 COSV56231737 4 TP50 M3 c.1447C > T p.(Gln483*) Nonsense Somatic Sanger 114,536,409 1 NA – TP51 M3 c.2057-4G > T exon 17 splice acceptor No effect Germ line Panel NA – NA – TP52 M3 c.2057-4G > T exon 17 splice acceptor No effect Germ line Sanger NA – NA – Mutation description refers to BAP1 transcript reference sequence NM_004656.3. In both tumors with partial loss of 3p regions, BAP1 gene is included in deleted region. With the exception of the variant indentified in TP7 all missense mutations are classified as oncogenic according to ACMG criteria as applicable Analysis of uveal melanomas and paired constitutional DNA for exclusion of a BAP1‑tumor… Fig. 2 Map of BAP1 oncogenic sequence variants in 50 uveal melanomas. a Black triangles: location of somatic missense or in-frame variants. b and c Black circles: location of premature stop codons due to nonsense, frameshift or splice variants caused by somatic altera- tions. Red circles: location of premature stop codons due to nonsense, frameshift or splice variants caused by constitu- tional alterations. Grey bar: in-frame loss of codons due to splice variant caused by somatic alteration. in-frame 1 729 BAP1 protein N-terminal UCH domain Key residues (Q82, C91, H169, D184) UCH37-like domain Coiled coil domain HCF1-binding domain NLS-motifs Fig. 3 Density plots of age at diagnosis of 140 patients included in the study grouped by chromosome 3 status Disomy 3 Monosomy 3Partial allele loss At the time of diagnosis of the primary tumor, data on cutaneous malignant melanoma. In the two other patients medical history concerning tumors outside of the eye was with diagnosis of cutaneous malignant melanoma prior to available from 100 patients. Two patients had a history of uveal melanoma, a common pathogenic BAP1 gene germline renal cell cancer diagnosed prior to uveal melanoma but only variant as root cause is highly unlikely because the BAP1 one of these two patients was heterozygous for a pathogenic gene mutations that we identified in their tumors were not BAP1 germ-line mutation. This patient also had a history of present in DNA from blood and, therefore, of somatic origin. 1 3 Age at diagnosis/years Y. A. Abbassi et al. However, as we cannot exclude low-dose mosaicism and In the tumors with partM3 the percentage of samples with thus it is conceivable that tumors can originate from cells of BAP1 mutation was much lower (27%) compared to M3 a mutant sector that share a oncogenic BAP1 gene somatic tumors which is in concordance with the observation that variant. Data on family history was incomplete for most most of these tumors show a mutation pattern typical for the patients. Of note, the father of a BAP1 heterozygous patient UM with D3 [20]. Between reported studies the proportion in our study had died from pleural mesothelioma, a tumor of tumors with M3 with a oncogenic BAP1 alteration varies type that is very rare in the general population but relatively and the detection rate in our study is well within this range frequent in families with BAP1-TPDS (for review see [14]). [23, 28]. It is conceivable that some tumors with M3 may have BAP1 gene alterations that are not within the scope of Discussion the detection methods used here. Specifically, we expect an improved rate of detection if sequencing libraries cover the In this study we used genetic testing of tumors for molecular complete BAP1 gene region instead of focusing enrichment differential diagnosis of BAP1-TPDS in patients with UM. of the exons only. The need for extension of the scope of We adapted a stepwise testing strategy similar to the one that analysis by NGS has already been pointed out by Field et al. is used for routine in genetic testing in retinoblastoma to the 2018 [23, 28]. Another advantage of NGS-based analysis is requirements specific for uveal melanoma. The initial analy - a lower detection limit compared to Sanger sequencing. This sis step identifies tumors with complete or partial monosomy allows the detection of sequence variants in heterogeneous 3 and, in the next step, DNA from these tumors is analyzed tumor tissue containing high proportion of normal cells [23] for BAP1 gene alterations. With decreasing sequencing costs thus increasing the proportion of fully informative cases, due to NGS, we would suggest sequencing of tumor and although it does not lead to the discovery of additional her- blood tissue simultaneously instead of one after another itable cases. which is more time efficient. A BAP1-TPDS is excluded In 2 patients (4% of 50 patients with BAP1 mutant tumors, in patients in whom a somatic origin of these alterations 1.4% of all 140 patients) we determined heterozygosity for is established by analysis of constitutional DNA. With this pathogenic BAP1 gene variants and this established the diag- three-step strategy, we could safely exclude a BAP1-TPDS nosis of a BAP1-TPDS. Both variants are expected to result in 50 of 72 (70%) patients with a M3 or partM3 UM. Since in premature termination which is the predominant type of germ-line variant alleles of the BAP1 gene are rare in UM consequence of pathogenic BAP1 alterations. Premature ter- patients with D3 tumors (odds ratio 0.12, derived from data mination was also the most frequent biological consequence published by Ewens 2018 [22]), TPDS is also unlikely in the among the BAP1 germ-line variants reported by Ewens et al. group of 62 patients with D3 tumors. However, it remains [22]. By contrast, studies restricted to mutation analysis on to be shown if extending the diagnostic strategy to patients DNA from blood tend to report a higher proportion of mis- with UM with D3 leads to detection of further BAP1-TPDS sense type variants and these include a high proportion of with a frequency higher than those in the normal (UM-free) variants of uncertain pathogenic significance (VUS) [22]. population [22]. In fact, 608 of 738 (82%) gene variants in the BAP1 coding Most of the alterations with confirmed somatic origin region listed in ClinVar in the context of heritable tumor resulted in premature termination outside of the regions susceptibility are VUS (https://www .ncbi. nlm. nih. go v/clin v that are expected to evade nonsense mediated mRNA decay ar, query of 14.7.2021). Using our strategy, which requires [26] and, therefore, are expected to result in loss of func- initial sequencing of tumor DNA, only half as much VUS tion, which is typical for the spectrum of BAP1 alterations are expected due to loss of one BAP1 allele in most tumors. in cancer. All amino acids altered by missense substitution In other words, results of mutation analysis on DNA from or short in-frame losses belonged to the ubiquitin C-terminal blood only are often insufficient to provide the evidence hydrolase (UCH) domain of the BAP1 protein. This catalytic needed to demonstrate or exclude a BAP1-TPDS with high domain plays a key role by mediating deubiquitination of confidence. In vitro assays that assess if some functions of chromatin elements such as H2A [27]. Most of the missense BAP1 protein are altered depending on certain alterations substitutions identified here have been reported as somatic can provide additional data in support of the pathogenicity mutations in other tumors and this also supports that these of exonic BAP1 variants [29, 30]. However, we agree that alterations can be classified as pathogenic. Recurrence of “further research into BAP1 functions is needed” to arrive somatic missense mutations was also found in a recent report at “a consensus on mandatory experimental assays to assess by Ewens et al. [22]. Thus, it appears that the set of onco- VUS” [30]. genic missense mutations of the BAP1 gene is smaller than Only one of the two patients that we identified to be the set of variants that result in premature termination. heterozygous for a pathogenic BAP1 alteration was also We did not identify oncogenic BAP1 alterations in DNA positive for features proposed as referral guidelines for from 14 to 8 tumors (29%) with M3 or partM3, respectively. genetic diagnostics of a BAP1-TPDS [31]. This patient 1 3 Analysis of uveal melanomas and paired constitutional DNA for exclusion of a BAP1‑tumor… Consent for publication All patients included in this study signed was diagnosed with CMM and RCC (at age 28 and informed consent regarding publishing their data. 39 years, respectively) prior to the diagnosis of UM (at age 59 years). Moreover, one parent of this patient had Open Access This article is licensed under a Creative Commons Attri- died of pleural mesothelioma, a rare cancer that is frequent bution 4.0 International License, which permits use, sharing, adapta- in patients with a BAP1-TPDS. The medical and family tion, distribution and reproduction in any medium or format, as long history of the other BAP1-TPDS patient identified in our as you give appropriate credit to the original author(s) and the source, study did not meet the referral criteria proposed by Chau provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are et al. [31]. However, with a relatively young age at diag- included in the article's Creative Commons licence, unless indicated nosis of UM (40 years) this patient qualifies according to otherwise in a credit line to the material. If material is not included in more relaxed guidelines [9]. From recent studies it appears the article's Creative Commons licence and your intended use is not that population-based analyses, which avoid the ascertain- permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a ment biases introduced by selection of high-risk patients, copy of this licence, visit http://cr eativ ecommons. or g/licen ses/ b y/4.0/ . are required to obtain a better understanding of the BAP1- TPDS [22]. In other cancer predisposition syndromes such as hereditable breast and ovarian cancer (HBOC) it References has turned out that initial estimates of penetrance, which were derived from observations of familial aggregation of 1. Kaliki S, Shields CL (2017) Uveal melanoma: relatively rare cancer cases, were inflated. Unbiased data are required to but deadly cancer. Eye (Lond) 31(2):241–257. https:// doi. org/ model the cancer risk of individuals who are heterozygous 10. 1038/ eye. 2016. 275 2. Krantz BA, Dave N, Komatsubara KM, Marr BP, Carvajal RD for a pathogenic BAP1 allele. Such a statistical model is (2017) Uveal melanoma: epidemiology, etiology, and treatment essential to derive surveillance plans for early detection. of primary disease. Clin Ophthalmol 11:279–289. https:// doi. The results of our study support that genetic diagnosis of org/ 10. 2147/ OPTH. S89591 a BAP1-TPDS should be offered to all patients with UM. 3. Weis E, Shah CP, Lajous M, Shields JA, Shields CL (2006) The association between host susceptibility factors and uveal Moreover, the testing strategy should include efforts to melanoma: a meta-analysis. Arch Ophthalmol 124(1):54–60. obtain genetic information from the tumor. https:// doi. org/ 10. 1001/ archo pht. 124.1. 54 4. Ferguson R, Vogelsang M, Ucisik-Akkaya E et al (2016) Genetic Acknowledgements We thank Prof. Dr. Hebebrandt for providing the markers of pigmentation are novel risk loci for uveal melanoma. nucleus for the initiation of the study. Sci Rep 6:31191. https:// doi. org/ 10. 1038/ srep3 1191 5. Lynch HT, Krush AJ (1968) Heredity and malignant mela- Author contributions Material preparation, data collection, and analy- noma: implications for early cancer detection. Can Med Assoc sis were performed by all authors. The first draft of the manuscript was J 99(1):17–21 written by Dietmar Lohmann and all authors commented on previous 6. Vogel F (1954) Genetics and mutation rate of retinoblastoma versions of the manuscript. All authors read and approved the final (glioma retinae), with general remarks on methods of determin- manuscript. DL: conceptualization; MZ, YAA: methodology; DL: for- ing mutation rate in humans. Z Mensch Vererb Konstitutionsl mal analysis and investigation; DL: writing—original draft preparation; 32(4):308–336 MZ: writing—review and editing; DL: funding acquisition. 7. Knudson AG Jr (1971) Mutation and cancer: statistical study of retinoblastoma. Proc Natl Acad Sci U S A 68(4):820–823. https:// doi. org/ 10. 1073/ pnas. 68.4. 820 Funding Open Access funding enabled and organized by Projekt 8. Wiesner T, Obenauf AC, Murali R et al (2011) Germline muta- DEAL. This work was supported by Deutsche Krebshilfe (Grant tions in BAP1 predispose to melanocytic tumors. Nat Genet No.70110960). 43(10):1018–1021. https:// doi. org/ 10. 1038/ ng. 910 9. Abdel-Rahman MH, Pilarski R, Cebulla CM et  al (2011) Data availability Not applicable. Germline BAP1 mutation predisposes to uveal melanoma, lung adenocarcinoma, meningioma, and other cancers. J Code availability Not applicable. Med Genet 48(12):856–859. https:// doi. or g/ 10. 1136/ jmedg enet- 2011- 100156 10. Han A, Purwin TJ, Aplin AE (2021) Roles of the BAP1 tumor Declarations suppressor in cell metabolism. Cancer Res 81(11):2807–2814. https:// doi. org/ 10. 1158/ 0008- 5472. CAN- 20- 3430 Conflicts of interest The authors have no relevant financial or non- 11. Carbone M, Harbour JW, Brugarolas J et al (2020) Biologi- financial interests to disclose. cal mechanisms and clinical significance of BAP1 mutations in human cancer. Cancer Discov 10(8):1103–1120. https://d oi.o rg/ Ethical approval This study was performed in line with the principles 10. 1158/ 2159- 8290. CD- 19- 1220 of the Declaration of Helsinki. Approval was granted by the Ethics 12. Carbone M, Ferris LK, Baumann F et al (2012) BAP1 cancer Committee of the University Hospital Essen (13–5462-B0). syndrome: malignant mesothelioma, uveal and cutaneous mela- noma, and MBAITs. J Transl Med 10:179. https:// doi. org/ 10. Consent to participate Written informed consent to participate was 1186/ 1479- 5876- 10- 179 given by every patient included in this study. 1 3 Y. A. Abbassi et al. 13. Testa JR, Cheung M, Pei J et  al (2011) Germline BAP1 melanoma. Nat Commun 9(1):116. https:// doi. or g/ 10. 1038/ mutations predispose to malignant mesothelioma. Nat Genet s41467- 017- 02428-w 43(10):1022–1025. https:// doi. org/ 10. 1038/ ng. 912 24. Betti M, Casalone E, Ferrante D et al (2015) Inference on ger- 14. Walpole S, Pritchard AL, Cebulla CM et al (2018) Comprehen- mline BAP1 mutations and asbestos exposure from the analysis sive study of the clinical phenotype of germline BAP1 variant- of familial and sporadic mesothelioma in a high-risk area. Genes carrying families worldwide. J Natl Cancer Inst 110(12):1328– Chromosomes Cancer 54(1):51–62. https:// doi. org/ 10. 1002/ gcc. 1341. https:// doi. org/ 10. 1093/ jnci/ djy171 22218 15. Star P, Goodwin A, Kapoor R et al (2018) Germline BAP1- 25. Carbone M, Pass HI, Ak G et al (2022) Medical and surgical positive patients: the dilemmas of cancer surveillance and a care of patients with mesothelioma and their relatives carrying proposed interdisciplinary consensus monitoring strategy. Eur germline BAP1 mutations. J Thorac Oncol 17(7):873–889. https:// J Cancer 92:48–53. https:// doi. org/ 10. 1016/j. ejca. 2017. 12. 022doi. org/ 10. 1016/j. jtho. 2022. 03. 014 16. Noorani HZ, Khan HN, Gallie BL, Detsky AS (1996) Cost com- 26. Lindeboom RG, Supek F, Lehner B (2016) The rules and impact parison of molecular versus conventional screening of relatives of nonsense-mediated mRNA decay in human cancers. Nat Genet at risk for retinoblastoma. Am J Hum Genet 59(2):301–307 48(10):1112–1118. https:// doi. org/ 10. 1038/ ng. 3664 17. Lohmann DR, Gerick M, Brandt B et al (1997) Constitutional 27. Okino Y, Machida Y, Frankland-Searby S, Machida YJ (2015) RB1-gene mutations in patients with isolated unilateral retino- BRCA1-associated protein 1 (BAP1) deubiquitinase antagonizes blastoma. Am J Hum Genet 61(2):282–294. https:// doi. org/ 10. the ubiquitin-mediated activation of FoxK2 target genes. J Biol 1086/ 514845 Chem 290(3):1580–1591. https:// doi. or g/ 10. 1074/ jbc. M114. 18. Tschentscher F, Husing J, Holter T et al (2003) Tumor clas- 609834 sification based on gene expression profiling shows that uveal 28. van de Nes JA, Nelles J, Kreis S et al (2016) Comparing the prog- melanomas with and without monosomy 3 represent two distinct nostic value of BAP1 mutation pattern, chromosome 3 status, and entities. Cancer Res 63(10):2578–2584 BAP1 immunohistochemistry in uveal melanoma. Am J Surg 19. Tschentscher F, Prescher G, Zeschnigk M, Horsthemke B, Lohm- Pathol 40(6):796–805. https:// doi. org/ 10. 1097/ PAS. 00000 00000 ann DR (2000) Identification of chromosomes 3, 6, and 8 aberra- 000645 tions in uveal melanoma by microsatellite analysis in comparison 29. Hong JH, Chong ST, Lee PH et al (2020) Functional characterisa- to comparative genomic hybridization. Cancer Genet Cytogenet tion guides classification of novel BAP1 germline variants. NPJ 122(1):13–17. https:// doi. org/ 10. 1016/ s0165- 4608(00) 00266-1 Genom Med 5:50. https:// doi. org/ 10. 1038/ s41525- 020- 00157-6 20. Martin M, Masshofer L, Temming P et al (2013) Exome sequenc- 30. Repo P, Jarvinen RS, Jantti JE et  al (2019) Population-based ing identifies recurrent somatic mutations in EIF1AX and SF3B1 analysis of BAP1 germline variations in patients with uveal mel- in uveal melanoma with disomy 3. Nat Genet 45(8):933–936. anoma. Hum Mol Genet 28(14):2415–2426. https:// doi. org/ 10. https:// doi. org/ 10. 1038/ ng. 26741093/ hmg/ ddz076 21. Richards S, Aziz N, Bale S et al (2015) Standards and guidelines 31. Chau C, van Doorn R, van Poppelen NM et al (2019) Families for the interpretation of sequence variants: a joint consensus rec- with BAP1-tumor predisposition syndrome in The Netherlands: ommendation of the American College of Medical Genetics and path to identification and a proposal for genetic screening guide- Genomics and the Association for Molecular Pathology. Genet lines. Cancers (Basel). https:// doi. org/ 10. 3390/ cance rs110 81114 Med 17(5):405–424. https:// doi. org/ 10. 1038/ gim. 2015. 30 22. Ewens KG, Lalonde E, Richards-Yutz J, Shields CL, Ganguly Publisher's Note Springer Nature remains neutral with regard to A (2018) Comparison of germline versus somatic BAP1 muta- jurisdictional claims in published maps and institutional affiliations. tions for risk of metastasis in uveal melanoma. BMC Cancer 18(1):1172. https:// doi. org/ 10. 1186/ s12885- 018- 5079-x 23. Field MG, Durante MA, Anbunathan H et  al (2018) Punc- tuated evolution of canonical genomic aberrations in uveal 1 3

Journal

Familial Cancer – Springer Journals

Published: Apr 1, 2023

Keywords: Uveal melanoma; BAP1 gene; BAP1-TPDS; Predictive testing

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