Patterns of BAP1 protein expression provide insights into prognostic significance and the biology of uveal melanoma

Abstract

Uveal melanoma (UM) is a rare aggressive intraocular tumour with a propensity for liver metastases, occurring in ∼50% of patients. The tumour suppressor BAP1 is considered to be key in UM progression. Herein, we present the largest study to date investigating cellular expression patterns of BAP1 protein in 165 UMs, correlating these patterns to prognosis. Full clinical, histological, genetic, and follow-up data were available for all patients. BAP1 gene sequencing was performed on a subset of 26 cases. An independent cohort of 14 UMs was examined for comparison. Loss of nuclear BAP1 (nBAP1) protein expression was observed in 54% (88/165) UMs. nBAP1 expression proved to be a significant independent prognostic parameter: it identified two subgroups within monosomy 3 (M3) UM, which are known to have a high risk of metastasis. Strikingly, nBAP1-positiveM3 UMs were associated with prolonged survival compared to nBAP1-negative M3 UMs (Log rank, p = 0.014). nBAP1 protein loss did not correlate with a BAP1 mutation in 23% (6/26) of the UMs analysed. Cytoplasmic BAP1 protein (cBAP1) expression was also observed in UM: although appearing 'predominantly diffuse' in most nBAP1-negative UM, a distinct 'focal perinuclear' expression pattern - localized immediately adjacent to the cis Golgi - was seen in 31% (18/59). These tumours tended to carry loss-of-function BAP1 mutations. Our study demonstrates loss of nBAP1 expression to be the strongest prognostic marker in UM, confirming its importance in UM progression. Our data suggest that non-genetic mechanisms account for nBAP1 loss in a small number of UMs. In addition, we describe a subset of nBAP1-negative UM, in which BAP1 is sequestered in perinuclear bodies, most likely within Golgi, warranting further mechanistic investigation.

Keywords: BAP1; Golgi; cytoplasm; immunohistochemistry; mutation; prognosis; uveal melanoma.

Figure 1

Prognostic value of nuclear BAP1 protein expression. (A–C) Representative images of nuclear BAP1 protein (nBAP1) expression in primary UM (x10 magnification); insets show nBAP1 at x40 magnification. (A) nBAP1 negative tumour with focal perinuclear cBAP1. Intrinsic nuclear positivity is present in the adjacent neural retinal tissue (bottom inset). (B) Positive (left) and negative (right) nBAP1 staining in pancreas control sections. (C) nBAP1 staining in non‐neoplastic uveal melanocytes. (D) nBAP1 positive UM (left) and a nBAP1 negative UM (right). (E, F) Kaplan‐Meier survival curves and tables for primary UM in the Liverpool cohort stratified according to: (E) nBAP1 protein expression (n = 165; Log Rank, p < 0.001) (nBAP1 protein expression was scored as positive or negative); or (F) chromosome 3 status (n = 152; Log Rank, p = 0.001), where only cases with a discernible normal or loss of chromosome 3 were included. Number of events indicates the number of deaths due to metastatic melanoma. Log‐rank tests were used to compare survival across groups. BAP1 positivity was detected using a brown chromogen in (A), (B), and (D) and a red chromogen in (C).

Figure 2

nBAP1 status contributes additional prognostic information in M3‐UM patients. (A) Pie chart summarizing the breakdown of nBAP1 staining and chromosome 3 status in the Liverpool cohort of patients, n = 165. (B, C) Kaplan‐Meier survival curves and tables estimating disease‐free survival in UM patients stratified by: (B) nBAP1+ve/–ve status in monosomy 3 (M3) patients, n = 104 and (C) nBAP1+ve/–ve status in disomy 3 (D3) patients, n = 48. Number of events indicates the number of deaths due to metastatic melanoma. Log‐rank tests were used to compare survival across groups.

Figure 3

Cytoplasmic BAP1 exhibits differential localisation in UM. (A) Images showing either diffuse (left; DAB) or focal perinuclear (centre; DAB and right; AEC) localisation of cytoplasmic BAP1 (cBAP1) in three nBAP1 negative UM (main image and insets are at ×10 and ×40 magnification, respectively; scale bars 200 μm). (B) Pie charts summarizing the breakdown of nBAP1 expression in the 127 UMs studied (left) and cBAP1 expression in the 59 nBAP1 negative cases (right). (C) Kaplan‐Meier survival curve for 54 nBAP1 negative UM patients stratified by focal perinuclear and diffuse cBAP1 staining patterns. The difference in survival was not significant (Log rank; p = 0.925). (D) Images showing similar localisation of focal perinuclear cBAP1 with the cis Golgi marker GM130 in 3 μm serial sections of two UM cases (I and II) (scale bars 60 μm).

Figure 4

UMs with focal perinuclear cBAP1 tend to harbour loss‐of‐function BAP1 mutations. (A) Schematic showing the location of mutations within the BAP1 gene. 5′ and 3′ UTRs are depicted in red. One UM had two mutations flanking exon 17 (+); (B) location of the mutations shown in (A) mapped to the BAP1 protein (UCH, ubiquitin carboxy‐terminal hydrolase domain; BARD1, BARD1‐binding domain; HBM, HCF1‐binding domain; BRCA1, BRCA1‐binding domain; ULD, UCH‐37‐like domain; NLS, nuclear localisation signal. The location of cysteine, histidine, and aspartic acid residues of the catalytic triad are shown in the catalytic domain. (C) Histogram showing the distribution of mutation types for the nBAP1 positive, nBAP1 negative (cBAP1 diffuse), nBAP1 negative (cBAP1 focal perinuclear), and nBAP1 negative (cBAP1 negative) UMs.