Comparison of Germline versus Somatic BAP1 Mutations for Risk of Metastasis in Uveal Melanoma

The BRCA1-associated protein 1 (BAP1) gene is located on chromosome 3p21 and encodes a nuclear localized, ubiquitin carboxy-terminal hydrolase tumor suppressor protein [1, 2]. BAP1 has been shown to bind to the BRCA1 protein enhancing BRCA1-mediated tumor suppression, and is involved in various biological processes including DNA damage response, regulation of the cell cycle and cell growth [2, 3]. Germline BAP1 mutations have been associated with hereditary predisposition to multiple different cancers that include uveal and cutaneous melanoma, malignant mesothelioma on exposure to asbestos, renal cell carcinoma and other cancer types, such as lung adenocarcinoma and meningioma, that are collectively referred to as BAP1 Tumor Predisposition Syndrome (BAP1-TPDS, OMIM #614327) [4, 5]. In vivo studies in melanocytic cells, have shown that BAP1 is involved in the maintenance of a melanocytic phenotype; the depletion of BAP1 protein levels result in dedifferentiation of cells and the acquisition of a more primitive, stem cell like phenotype [6]. It is well established that the BAP1 region of chromosome 3p is commonly deleted in several cancers including melanomas, breast and lung cancers, among others [1, 7].

Uveal melanoma (UM) is a rare malignant tumor of the eye with a frequency of 5.1 per million in the United States [8], and is the cancer most commonly associated with the BAP1-TPDS [9]. The prognosis for UM is poor in approximately 50% of cases primarily due to metastasis to the liver within a short time [10, 11]. Factors such as large tumor size, location and chromosome 3 monosomy [12‐18], as well as a specific 12-gene expression pattern [19‐21] have all been associated with an increased risk of metastasis. In addition, the presence of inactivating somatic [22‐29] or germline [30] BAP1 mutations often in conjunction with chromosome 3 monosomy, loss of BAP1 expression, or lack of immunohistochemical staining, have all been associated with metastasizing UM.

Germline BAP1 mutations have been identified in approximately 2-5% of UM unselected for the presence of any high risk of hereditary cancers [30‐33]. These studies reported that metastasis occurred more frequently in UM carrying germline mutations in the blood compared to BAP1 mutation-negative controls, but the differences were significant only in one study [30], possibly due to the small number of tumors with germline mutations. In studies where DNA from both tumor tissue and blood was sequenced to rule out the presence of germline mutations, the frequency of somatic BAP1 mutations was considerably higher than that observed in tumors with germline mutations, approaching 50% [23, 27, 34]. In other studies where somatic mutations were detected by BAP1 expression assays and/or negative immunohistochemical staining methods, as well as sequencing, the lack of BAP1 expression was also significantly associated with the risk of metastasis [22‐29]. While these studies show a strong association between somatic BAP1 mutations and metastasis, the effect of germline changes remains unclear.

Most studies of BAP1 mutation status in UM have been carried out using enucleated UM which may not be representative of how UM are currently treated, especially smaller tumors that are treated with globe sparing procedures. In this study of BAP1 mutations in UM, the presence of germline or somatic mutations was determined using sequence analysis of DNA extracted from biopsies of both enucleated globes and fine needle aspirations (FNA) and from matched blood samples. Demographic and clinical characteristics of the tumors and the frequency of metastasis were compared among tumors with germline or somatic BAP1 mutations and mutation-negative UM.

Inactivating somatic mutations in the BAP1 gene in UM were first identified and associated with metastatic disease in 2010 [23]. Subsequent to this original publication, there have been numerous studies characterizing BAP1 somatic [22, 24‐29, 40‐43] and germline [9, 30, 32, 33] mutations in UM. However, to our knowledge, this is the first report directly comparing risk of metastasis in tumors carrying germline or somatic BAP1 mutations and mutation-negative tumors. In this study, the presence of somatic mutations was strictly defined as those found in tumors, and not in matched blood samples. A second important difference in this study is that, compared to most other reports where UM tumors analyzed for BAP1 mutations were comprised primarily of samples biopsied from larger, enucleated tumors, 84% of the samples analyzed in this study were from FNA biopsies. We found no significant difference in the frequency of somatic and germline mutations in biopsies from FNAs compared to enucleated tumors. Since the majority of UM are currently being treated by globe sparing procedures, it was important to determine whether the consequences of carrying a BAP1 mutation identified in larger enucleated tumors could be applied to smaller UM biopsied by FNA sampling.

It is well established that tumor characteristics such as tumor diameter, ciliary body involvement and chromosome 3 monosomy are all associated with poor prognosis as measured by the development of metastases within 48 months after the primary is treated [12‐18]. It has also been shown that that these same variables are significantly associated with BAP1 mutation status in tumors with somatic or germline mutations [24, 25, 28, 30]. In this study, multivariate regression analysis showed that in addition to these classic tumor variables, the presence of somatic, but not germline, BAP1 mutations was significantly associated with metastasis (HR=2.20, P=0.02; HR=0.87, P=0.81, respectively). Kaplan-Meier analysis further showed that tumors with somatic mutations were associated with the poorest metastatic outcome, while there was no significant difference between tumors carrying a germline mutation and mutation-negative tumors. Furthermore, the time to metastasis was seven months shorter for individuals whose tumors carried somatic mutations compared to mutation-negative tumors (P=0.04) suggesting a more aggressive phenotype. Thus, in a previous publication [37] we showed that tumors with any mutation in the BAP1 gene, in combination with chromosome 3-monosomy, have the highest risk of metastasis (HR=11.5). In this manuscript we show that monosomy 3 plus a somatic mutation in the BAP1 gene, as opposed to a germline mutation, have a significantly higher risk of metastasis (P=0.01, H.R.=2.81) and a shorter time to metastasis compared to the ones with a germline mutation.

Given that somatic BAP1 mutations appear to have a highly significant association with poor metastatic prognosis, it is surprising that the age of diagnosis of the individuals whose tumors carried somatic mutations was significantly older (median=61.8 years) compared to those with mutation-negative tumors (median=52.2 years, P=0.002). Previous studies have also reported that tumors with somatic mutations were diagnosed at least 8 years later than mutation-negative controls [24, 27, 29], but the significance of this observation is not clear.

Among the 54 tumors with BAP1 mutations, there were four with chromosome 3 disomy. It cannot be ruled out that although these tumors appeared to be disomy-3, small deletions not detectable on the SNP arrays leading to partial monosomy or heterogeneity of the tumor sample could be responsible for this finding. Alternatively, in some cases it is possible the during tumor evolution, BAP1 mutation on one copy of chromosome 3 could precede the loss of the other copy of chromosome 3. While several studies have found total concordance between the presence BAP1 mutations and monosomy 3 [23, 27, 40], others using sequencing, gene expression, and/or immunostaining methods have identified BAP1 mutations in a small number of tumors with disomy 3 [24, 25, 28, 44].

One interesting observation in this study was the presence of BAP1-TPDS history in 44% of somatic cases and 23% of mutation negative tumors. The data on BAP1-TPDS was based on self-reported personal and family history of cancer. We classified individuals as having BAP1-TPDS if they carried at least two relevant tumors in their personal or family history. Since it is self-reported, the information can be imprecise, and there is a possibility for over/under representation of the BAP1-TPDS cases as well.

While the presence of germline BAP1 mutations in cancers associated with BAP1-TPSD is well documented, the role of somatic mutations in this syndrome is not well studied. It is possible that the presence of TPDS in our cohort of UM cases that have a somatic BAP1 mutation or are mutation-negative could be interpreted as evidence for the existence of a second type of the syndrome associated with mutations in a second gene different from BAP1.

This study identified significant differences in the risk of metastasis for individuals whose tumors carry somatic vs. germline BAP1 mutations. While overall 36 of 59 (61%) metastasizing tumors carried a BAP1 mutation, only 7% carried germline mutations, compared to 54% with somatic mutations. From the viewpoint of counseling individuals with UM, there are different implications depending on whether the mutation is classified as germline or somatic. The most relevant information for the individuals with UM is the risk of metastasis, as well as an estimate of metastasis-free survival. The presence of germline mutations can provide the individual with information concerning the risk of development of other cancers themselves or in other family members, while information about the presence of somatic mutations is relevant to their individual risk of developing metastases within a shorter time period. Thus it is important to determine whether a tumor carries a BAP1 mutation, and if positive, to also evaluate a matched blood sample to establish whether the mutation is germline or somatic. In addition, this information can also change the cascade screening and surveillance of the family members.