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Table of Contents
ORIGINAL ARTICLE
Year : 2020  |  Volume : 2  |  Issue : 1  |  Page : 26

Expression of BRCA1-associated protein 1 in primary tumors and metastasis in an animal model of uveal melanoma


1 McGill University Health Centre, Research Institute, The MUHC – McGill University Ocular Pathology and Translational Research Laboratory, Montréal, Canada; Escola de Medicina e Cirurgia, Universidade Federal do Estado do Rio de Janeiro – UNIRIO, Rio de Janeiro, Brazil
2 McGill University Health Centre, Research Institute, The MUHC – McGill University Ocular Pathology and Translational Research Laboratory, Montréal, Canada

Date of Submission23-Jun-2020
Date of Acceptance03-Jul-2020
Date of Web Publication18-Sep-2020

Correspondence Address:
Ms. Clara E Castro
Rua Campos Novos 74/103, Barra Da Tijuca. Rio De Janeiro-RJ 22620-400

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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/PAJO.PAJO_29_20

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  Abstract 


Background: Uveal melanoma (UM) is the most common primary intraocular tumor in adults. Despite successful local treatment, approximately 50% of these patients develop lethal metastatic disease. BRCA1-associated protein 1 (BAP1) encodes BAP1, which functions as a tumor suppressor. Inactivation by a somatic mutation in BAP1 results in a loss of protein expression, which is associated with increased risk of metastasis.
Aim: To analyze the expression of BAP1 protein in primary uveal tumors and their corresponding metastasis in an animal model of UM and to determine whether BAP1 expression is lost during the metastatic process.
Subjects and Methods: Postmortem eyes and lungs with metastasis from nine rabbits previously inoculated with human UM cells expressing BAP1 (92.1 strain) were analyzed. Immunohistochemistry was performed on formalin-fixed paraffin-embedded samples using a monoclonal BAP1 (C-4) antibody. Expression was evaluated by the extent and intensity of staining. All primary tumors and metastasis were positive for BAP1.
Results: The staining was diffuse and intense in the primary tumors of four rabbits (67%), focal-intense in one rabbit and focal-mild in one rabbit. All lung metastasis showed focal staining. All animals with intense BAP1 staining in the primary tumor showed mild BAP1 in the metastasis.
Discussion: A predominant pattern observed between primary and metastatic lesion shows a transition from moderate into mild intensity and diffuse into the focal extent, suggesting a decrease in BAP1 expression. Future studies, including the human tissue of primary UM and their corresponding metastasis, should be performed to corroborate these findings.

Keywords: Animal model, BRCA1-associated protein 1 mutation, immunohistochemistry, metastasis, uveal melanoma


How to cite this article:
Castro CE, Burnier JV, Bergeron S, Coblentz J, Dias AB, Burnier MN. Expression of BRCA1-associated protein 1 in primary tumors and metastasis in an animal model of uveal melanoma. Pan Am J Ophthalmol 2020;2:26

How to cite this URL:
Castro CE, Burnier JV, Bergeron S, Coblentz J, Dias AB, Burnier MN. Expression of BRCA1-associated protein 1 in primary tumors and metastasis in an animal model of uveal melanoma. Pan Am J Ophthalmol [serial online] 2020 [cited 2020 Oct 21];2:26. Available from: https://www.thepajo.org/text.asp?2020/2/1/26/295337




  Introduction Top


Uveal melanoma (UM) is the most common primary intraocular tumor in adults, primarily found in the Caucasian population with a mean age-adjusted incidence of 5.1 cases per million per year in the United States.[1] UM can develop asymptomatically and is usually diagnosed by routine ophthalmic examination. It can cause painless distortion of vision and other nonspecific visual symptoms.[2] Currently, the most widely used first-line treatments for this malignancy are tumor resection, local radiation therapy, and enucleation.[1] Despite successful local treatment, approximately 50% of these patients develop lethal metastatic disease, which usually involves the liver and is rarely detectable at the time of treatment of the primary UM, developing varyingly months to years after diagnosis.[3] The mean survival after the diagnosis of metastatic disease is approximately 1 year, as limited therapeutic options are available once the disease has disseminated.[2],[4]

A strong predictor for metastasis is the loss of chromosome 3.[4] Inactivating mutations in BRCA1-associated protein 1 (BAP1), located on chromosome 3p21.1 and encoding the BAP1, have been reported in predominantly metastasizing UM.[4]BAP1 is a deubiquitinating enzyme which functions as a tumor suppressor protein through its deubiquitinase activity that regulates target genes in cell cycle control, cellular differentiation, and DNA damage repair.[5] Therefore, inactivation of BAP1 results in a loss of protein expression, which is associated with an increased risk of metastasis and low survival rate.[2] It is important to investigate the role of BAP1 and other deubiquitinating enzymes as potential therapeutic targets in metastatic cancer.[6]

The aim of this study is to analyze the presence of BAP1 protein expression in primary uveal tumors and their corresponding metastasis in an animal model of UM and to determine whether BAP1 is lost during the metastatic process. Immunohistochemistry (IHC) was performed to assess whether there is a correlation in the presence of BAP1 protein between primary and metastatic UM and to further validate the relevance of BAP1 IHC as part of the routine workup for UM patients.


  Subjects and Methods Top


The animal model was carried out in compliance with the Institutional Research Ethics Board of the Research Institute of the McGill University Health Centre (Protocol 2016–7629).

This is a retrospective study using postmortem eyes and lungs with metastasis from nine female New Zealand albino rabbits (Charles River Canada, St-Constant, Québec, Canada) that were inoculated with human UM cells expressing BAP1 (92.1 strain).[7] The cells were injected in the suprachoroidal space of the right eye for each rabbit as previously described, and tumor progression was monitoredin vivo for 10 weeks.[8] At the experimental endpoint, animals were euthanized, and organs were harvested for histopathological analysis.

Formalin-fixed paraffin-embedded (FFPE) sections for the eyes and lung of each rabbit were obtained for IHC, and representative sections were stained with hematoxylin and eosin (H and E) for histopathological assessment.

IHC was performed on FFPE sections using the Ventana Benchmark Automated Platform (Ventana Medical Systems, USA). A monoclonal BAP1 (C-4) antibody (Santa Cruz Biotechnology, USA, sc-28383) was applied at a dilution 1:2000. Slides were digitized using the Zeiss Axio Scan. Z1 Scanner (Zeiss, Germany). Expression was evaluated by extent and intensity of staining. Extent was scored as either focal or diffuse, and intensity was scored as mild or moderate.


  Results Top


All primary tumors and metastasis were positive for BAP1. Three rabbits were excluded from our dataset due to insufficient material. As per the BAP1 WT status of the 92.1 cells inoculated in the animals, all primary uveal tumors were BAP1 positive. The staining was diffuse and moderate in four rabbits (67%) [Figure 1]a and [Figure 1]b, focal-moderate in one rabbit (17%) [Figure 2]a and focal-mild in one rabbit (17%). Conversely, all lung metastasis showed focal staining, independently of the primary tumor. Four metastasis (67%) showed mild staining and two metastasis (33%) showed moderate staining, of which only one showed to be more intense than the primary tumor or the normal lung parenchyma. All animals with intense BAP1 staining in the primary tumor showed mild BAP1 in the metastasis [Figure 1]c and [Figure 2]b. It was not possible to determine whether the staining was nuclear or cytoplasmic due to staining quality.
Figure 1: BRCA1-associated protein 1 immunohistochemistry of an intraocular tumour and its corresponding lung metastasis. (a) BRCA1-associated protein 1 immunohistochemistry in an intraocular tumour showing two distinctives cell populations; one negative and one positive with moderate intensity and diffuse extent. (b) Higher magnification (×10) image of the intraocular tumour showing large epithelioid cells with mild and moderate diffuse staining. (c) Representative high magnification (×10) image of the corresponding lung metastasis (BRCA1-associated protein 1 mild) with inflammatory infiltrate (BRCA1-associated protein 1 positive)

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Figure 2: BRCA1-associated protein 1 immunohistochemistry of intraocular tumour and its corresponding lung metastasis. (a) Low power light (×1 magnification) microscopy image of an intraocular tumour with extraocular involvement. The neoplastic process is diffusely positive with high intensity of staining. The extraocular portion of this tumour shows a similar pattern in the periphery and a low expression of BRCA1-associated protein 1 in the center of the mass. (b) High-magnification (×10) image of lung metastasis from the same rabbit showing normal lung tissue expressing BRCA1-associated protein 1 and a small metastatic process showing only mild BRCA1-associated protein 1 staining (arrow)

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The distribution of the evaluation of BAP1 expression in primary tumors and metastasis, according to extent and intensity, is demonstrated in [Figure 3].
Figure 3: Graphical representation of the expression of BAP1 in primary tumors (eye) and metastasis (lung), according to extent and intensity. Extent is scored as either focal or diffuse and intensity is scored as mild or moderate. Graphical summary: Eye lung moderate 5 2 Mild 1 4 eye lung diffuse 4 0 Focal 2 6

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  Discussion Top


Somatic mutations in BAP1 result in a loss of protein expression, which is associated with an increased risk of metastasis and impart low survival rate.[2] To the best of our knowledge, while BAP1 mutations have been extensively studied in UM, this is the first study to investigate the potential loss of BAP1 in a primary UM tumor and its metastasis using a rabbit animal model.

Metastasis from the lung was chosen as in this animal model, the lung was the primary site for metastasis. Hepatic micrometastasis was also found to a lesser extent. This finding is noteworthy because liver metastasis has not yet been reported using this particular experimental model or similar ones and most important because the liver is the preferential site of metastasis in human UM.[8]

A pattern of decreased expression was seen from the primary (eye) to the metastatic lesion (lung) in most animals. There was a transition from moderate into mild intensity and diffuse into focal extent, suggesting a decrease in BAP1 expression. This is in accordance with the loss of BAP1 expression seen in most human UM metastatic tumors.[5] Due to staining, quality is was not possible to determine the localization of BAP1 in the cell, which would be important as localization to the nucleus is required for BAP1-mediated tumor suppression.[9]

Analysis of the loss of BAP1 expression during disease progression is pertinent, as its inactivation could potentially be a key player in the development of metastasis. Moreover, BAP1 and other deubiquitinating enzymes are prospective therapeutic targets in cancer and could be used to treat UM.[6]

As this is a retrospective study based on material obtained from a previous study, one of the main limitations was the quantity of material available for analysis.[8] Future studies, including human tissue of primary UM and their corresponding metastasis, should be performed to corroborate these findings. In addition, genomic profiling of the tissue DNA could validate the presence of loss-of-function mutations. It is also important to note that, even though a strong correlation has been reported between BAP1 IHC and sequencing data, the use of IHC cannot, in itself, confirm the BAP1 gene status (WT or mutated).[4] Furthermore, UM metastasizes predominantly to the liver, and not the lungs, in humans; therefore, our findings need verification and validation in a liver-metastatic model and in human tissue.

Our observations support a loss of BAP1 expression in metastatic UM. This prompts the importance of adding BAP1 status to the routine workup of UM patients and suggests a possible change of BAP1 status during the metastatic process.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
  References Top

1.
Kaliki S, Shields CL. Uveal melanoma: Relatively rare but deadly cancer. Eye (Lond) 2017;31:241-57.  Back to cited text no. 1
    
2.
Amaro A, Gangemi R, Piaggio F, Angelini G, Barisione G, Ferrini S, et al. The biology of uveal melanoma. Cancer Metastasis Rev 2017;36:109-40.  Back to cited text no. 2
    
3.
Kalirai H, Dodson A, Faqir S, Damato BE, Coupland SE. Lack of BAP1 protein expression in uveal melanoma is associated with increased metastatic risk and has utility in routine prognostic testing. Br J Cancer 2014;111:1373-80.  Back to cited text no. 3
    
4.
Koopmans AE, Verdijk RM, Brouwer RW, van den Bosch TP, van den Berg MM, Vaarwater J, et al. Clinical significance of immunohistochemistry for detection of BAP1 mutations in uveal melanoma. Mod Pathol 2014;27:1321-30.  Back to cited text no. 4
    
5.
Masoomian B, Shields JA, Shields CL. Overview of BAP1 cancer predisposition syndrome and the relationship to uveal melanoma. J Curr Ophthalmol 2018;30:102-9.  Back to cited text no. 5
    
6.
Harbour JW, Onken MD, Roberson ED, Duan S, Cao L, Worley LA, et al. Frequent mutation of BAP1 in metastasizing uveal melanomas. Science 2010;330:1410-3.  Back to cited text no. 6
    
7.
Zhao H, Darden J, Chappell JC. Establishment and characterization of an embryonic pericyte cell line. Microcirculation 2018;25:e12461.  Back to cited text no. 7
    
8.
Blanco PL, Marshall JC, Antecka E, Callejo SA, Souza Filho JP, Saraiva V, et al. Characterization of ocular and metastatic uveal melanoma in an animal model. Invest Ophthalmol Vis Sci 2005;46:4376-82.  Back to cited text no. 8
    
9.
Ventii KH, Devi NS, Friedrich KL, Chernova TA, Tighiouart M, Van Meir EG, et al. BRCA1-associated protein-1 is a tumor suppressor that requires deubiquitinating activity and nuclear localization. Cancer Res 2008;68:6953-62.  Back to cited text no. 9
    


    Figures

  [Figure 1], [Figure 2], [Figure 3]



 

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