UVEAL MELANOMA: GENETIC AND EPIGENETIC CHARACTERISATION
Charlotte L. Ness MD
Institute of Clinical Medicine, Faculty of Medicine, University of Oslo
and
Department of Ophthalmology, Oslo University Hospital
Ph.D. thesis 2020
© Charlotte Larsen Ness, 2021 Series of dissertations submitted to the Faculty of Medicine, University of Oslo
ISBN 978-82-8377-853-3
All rights reserved. No part of this publication may be
reproduced or transmitted, in any form or by any means, without permission.
Cover: Hanne Baadsgaard Utigard.
Print production: Reprosentralen, University of Oslo.
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TABLE OF CONTENTS
Contents
1. Acknowledgements 4
2. Abbreviations 5
3. List of papers 7
Paper I 7
Paper II 7
Paper III 7
4. Sammendrag 8
5. Introduction 10
5.1 Uveal melanoma. Disease and management 10
5.1.1 Location, epidemiology and risk factors 10
5.1.2 Symptoms and diagnosis 11
5.1.3 TNM classification and prognostic pathological parameters 12
5.1.4 Treatment 13
5.1.5 Management of metastatic disease 14
5.2 Genetic determinants in uveal melanoma 15
5.2.1 Cytogenetic features 15
5.2.2 Molecular pathways and genomic alterations in UM 15
5.2.3 Binary clustering of uveal melanomas 20
5.2.4 Genetic alterations in metastatic UM 23
5.3 Epigenetics 23
5.3.1 DNA methylation 24
5.4 In vitro and in vivo preclinical models for studying UM 27
5.4.1. Three-dimensional in vitro models 28
5.4.2 In vitro assays for studying the metastatic process of UM 29
5.4.3 In vivo assays for studying UM 30
6. Aims of the thesis 31
7. Methods and methodological considerations 32
7.1 In vitro cultivation 32
7.2 Immunohistochemistry 33
7.3 Electron microscopy 35
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7.4 Real-time quantitative reverse transcription PCR (qRT-PCR) 36
7.5 RNAscope in situ hybridisation 38
7.6 Microarrays 39
7.6.1 cDNA microarrays 39
7.6.2 DNA methylation assay 41
7.7 Western blot 43
8. Summary of results 44
8.1 Paper I 44
8.2 Paper II 44
8.3 Paper III 45
9.1 Discussion Paper I 47
9.2 Discussion Paper II 49
9.3 Discussion Paper III 52
10. Conclusions and future perspectives 55
11. References 57
Paper I-III 76
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1. Acknowledgements
The work leading to this thesis was performed at Center for Eye Research, Department of Ophthalmology, Oslo University Hospital and University of Oslo.
I wish to thank everyone who has contributed to this thesis. In particular I would like to express my deepest gratitude to co-supervisor Agate Noer without whom this thesis would not have been completed. Your guidance, extensive knowledge and the amount of time you have put in the project has been of immense importance. Thank you for being optimistic and encouraging throughout these years.
I would like to extend my sincere thanks to my supervisor, Professor Morten C. Moe for offering me the opportunity to carry out my PhD work and for constructive feedback and support. I am also grateful to my co-supervisor Professor Emeritus Bjørn Nicolaissen for his valuable advises.
My gratitude goes to Kirankumar Katta for his indispensable work on paper III. Special thanks to Øystein Garred and Theresa Kumar for providing in-depth histopathological knowledge and help in obtaining tissue samples. Further, I would like to acknowledge the contributions of all co-authors.
Many thanks go to my colleagues at Center for Eye Research for practical support and for creating a social work environment.
Lastly, my appreciation goes to close friends and family for their patience and encouragement.
The work was funded by the South-Eastern Norway Regional Health Authority (project 2012104), Norwegian Cancer Society (project 5808589) and supported by grants from the Norwegian Association of the Blind and Partially Sighted, Arthur and Odd Clausons ophthalmological fund, Aase and Knut Tønjums ophthalmological fund, Futura fund, Unifor Frimed, Inger Holms memorial fund, “Stiftelsen for fremme av kreftforskning” at University of Oslo and “Legat til fremme av kreftforskning”.
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2. Abbreviations
AEC aminoethyl carbazole
AR antigen retrieval
BAP1 BRCA1 Associated Protein 1
BRAF B-RAF Proto-Oncogene
BSA bovine serum albumin
CNV copy number variation
CpG 5`-cytosine-phosphate-guanine-3
CPGI 5`-cytosine-phosphate-guanine-3` island
CSC cancer stem cell
CT computed tomography
Ct threshold cycle
CTL4 cytotoxic T lymphocyte antigen 4
Cq quantification cycle
Cx43 connexin 43
DAB 3,3`-diaminobenzidine
DMR differentially methylated region DMP differentially methylated position
DNA deoxyribonucleic acid
DNMT DNA methyltransferase ECM extracellular matrix
EZH2 enhancer for zeste homolog 2 FAO fatty acid oxidation
FFPE formalin-fixed paraffin embedded FITC fluorescein isothiocyanate
FNAB fine needle aspiration biopsy
GNA11 guanine nucleotide-binding protein subunit alpha-11 GNAQ guanine nucleotide-binding protein G(q) subunit alpha HIER heat induced epitope retrieval
HRP horseradish peroxidase
ICI immune checkpoint inhibitors
IHC immunohistochemistry
6 IPA ingenuity pathway analysis
LOA loss of adherence
M3 monosomy 3
MCTS multicellular tumour spheroids
MEK mitogen-activated protein kinase kinase MRI magnetic resonance imaging
PCA principal component analysis PCR polymerase chain reaction PD1 programmed cell death protein 1 PET positron emission tomography PRC1 polycomb repressive complex 1 PRC2 polycomb repressive complex 2
PR-DUB polycomb repressive-deubiquitinase complex
qRT-PCR quantitative reverse transcription polymerase chain reaction
RIN RNA integrity number
RNA ribonucleic acid
RPE retinal pigmented epithelium SEM scanning electron microscope TEM transmission electron microscope TET ten-eleven translocation enzymes tRNA transfer ribonucleic acid
TSS transcription start site
UM uveal melanoma
2D two-dimensional
3D three-dimensional
5-hmC 5-hydroxymethylcytosine
5-mC 5-methylcytosine
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3. List of papers
Paper I
C. Ness, Ø. Garred, N. Eide., T. Kumar, OK. Olstad, TP. Bærland, G. Petrovski, MC. Moe, A.
Noer
Multicellular tumor spheroids of human uveal melanoma induce genes associated with anoikis resistance, lipogenesis, and SSXs.
Exp Eye Res. 2021 Feb;203:108426. doi: 10.1016/j.exer.2020.108426. Epub 2020 Dec 30.
Paper II
C. Ness, K. Katta, Ø. Garred, T. Kumar, OK. Olstad, G. Petrovski, MC. Moe. A. Noer
Integrated differential DNA methylation and gene expression of formalin-fixed paraffin- embedded uveal melanoma specimens identifies genes associated with early metastasis and poor prognosis
Mol Vis. 2017 Oct 3;23:680-694. eCollection 2017.
Paper III
K. Katta*, C. Ness*, Ø. Garred, T. Kumar, OK. Olstad, N. Eide, B. Nicolaissen, G. Petrovski, MC. Moe. A. Noer
*= co-first authors
Connexin 43 expression and subcellular distribution is dysregulated in human uveal melanoma
Manuscript
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4. Sammendrag
Uvealt malignt melanom (UM) er den vanligste formen for primær øyekreft. Det er en alvorlig sykdom hvor opptil 50% av pasientene utvikler metastaser. Ved spredning har en ingen effektive behandlingsalternativer. Avhandlingen består av 3 separate arbeider hvor en undersøker basale mekanismer ved UM som kan ha betydning for spredning og som kan representere angrepspunkter for fremtidig behandling.
Den første artikkelen tar for seg mekanismer som kan være assosiert med disseminering av kreftceller og overlevelse av de disseminerte cellene. En sammenlignet i dette arbeidet primærtumorer mot adherente cellekulturer og multicellulære-tumorsfæroider (MCTS).
Effekten av de forskjellige vekstbetingelsene ble undersøkt vha. elektronmikroskopi, DNA- mikromatrise, qRT-PCR, RNAscope og immunohistokjemi (IHC). MCTS fremviste egenskaper assosiert med motstand mot anoikis, som oppregulering av ANGPTL4 og økt fettmetabolisme. MCTS viste også økt ekspresjon av Synovial sarcoma, X breakpoint proteiner (SSXer), SSXer er kjente mål for immunterapi ved andre kreftformer.
I den andre artikkelen undersøkte en DNA-metyleringsmønstre i UM. Endringer i DNA- metylering har betydning for utvikling og progresjon av kreft. Disse endringene er i utgangspunktet reversible og er derfor attraktive mål for kreftbehandling. En undersøkte i denne studien DNA-metylering i eldre formalinfikserte parafinblokker hvor en koblet funn til data fra patologirapporter, Kreftregisteret og Dødsårsaksregisteret. DNA-metylering ble analysert vha.
Illumina Infinium HumanMethylation450 mikromatrise. En utførte videre en integrert analyse mellom DNA-metylering og genekspresjon på et utvalg av prøver og fant her endringer assosiert med tidlig metastasering. Genuttrykket ble undersøkt vha. DNA-mikromatrise og qRT-PCR. Hypermetylerte gen inkluderte de antatte tumorsuppressor-genene RNF13, ZNF217 og HYAL1, mens hypometylerte gen inkluderte de antatte onkogenene TMEM200C, RGS10, ADAM12 og PAM.
I det tredje arbeidet undersøkte en ekspresjon av connexin 43 (Cx43) i primære UM og vurderte effekten av inhibering av Enhancer of Zeste homolog 2 (EZH2) på Cx43-ekspresjon.
Connexiner er involvert i en rekke cellulære prosesser og er ofte dysregulerte ved kreft. UM biopsier og cellekulturer ble sammenlignet med choroidale biopsier og uveale melanocytter fra friske donorer vha. DNA-mikromatrise og qRT-PCR. Cx43-uttrykk i primærtumorer ble undersøkt vha. IHC og korrelert med histopatologiske data. Videre undersøkte en effekten av EZH2-inhibitoren Tazemetostat på Cx43-ekspresjon i UM cellelinjer. Effekten ble evaluert vha. morfologisk vurdering, ATP-analyse, qRT-PCR, immunocytokjemi (ICC) og Western
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blot. Ekspresjon av Cx43 var redusert i UM i forhold til friske kontroller. UM fremviste også redusert membran-innfarging. Tazemetostat medførte ikke endringer i Cx43-ekspresjon, men en observerte en reduksjon av H3K27me3 uavhengig av BAP1 status.
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5. Introduction
Cancer is a leading cause of death worldwide and can be looked upon as diverse collection of diseases characterised by the dysregulation of important pathways that control normal cellular homeostasis (1). In 2000 Hanahan and Weinberg presented six hallmarks of cancer as a framework to explain the escape from normal control mechanisms. The six hallmarks include sustaining proliferative signalling, evading growth suppressors, resisting cell death, enabling replicative immortality, inducing angiogenesis and metastasis (2). In 2011 two new hallmarks were added, namely the reprogramming of energy metabolism and the evasion of immune destruction (3). Factors leading to these hallmarks include both genetic and epigenetic events.
Uveal melanoma (UM) is a relatively rare malignancy, thus receiving far less research attention than cancers with a higher incidence. Compared to cutaneous melanoma, the unravelling of the biology of UM is still in its beginning. UM has a high propensity for metastatic spread and is a devastating disease for 50% of the patients (4). The lack of effective therapeutic options for patients with metastatic disease urges the need for in-depth biological knowledge in order to develop new and improved therapeutics.
5.1 Uveal melanoma. Disease and management
5.1.1 Location, epidemiology and risk factors
UM is the most common primary intraocular malignancy with an incidence of 5-8 cases per million per year in Norway (4). The tumour arises from the pigmented cells of the posterior uveal tract (choroid and ciliary body) or the anterior uveal tract (iris) (Figure 1). The choroid is the most common location comprising approximately 85% of the cases. Iris melanomas constitute 5% of the tumours, while ciliary body melanomas comprise 10% of the cases (4, 5).
The incidence is higher for those of Caucasian ethnicity, and especially for individuals with fair skin and light iris colour. In contradistinction to skin melanoma, UM is not evidently associated with sun exposure (6, 7). The median age at diagnosis is 60 years for patients with tumours of the choroid and ciliary body, while the median age for iris melanomas is lower, at 43 years (8, 9). A significant predilection for gender has not been shown (4). Choroidal nevi can in some instances undergo malignant transformation (10).
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Approximately 40-50% of the patients develop metastatic disease. Once metastatic disease occurs, the survival rate drops dramatically with a life expectancy of only 4 -17 months (11).
Systemic metastases are most commonly found in the liver (89%), followed by lungs (29%), bone (17%) and skin (12%). An estimated 10% of the patients develop another primary malignancy (12).
Figure 1: Anatomy of the eye. Horizontal section. (Courtesy of Geir A. Qvale, Oslo, Norway)
5.1.2 Symptoms and diagnosis
Initially most of the tumours are asymptomatic, though some patients can experience early symptoms secondary to the localisation of the tumour. Iris melanomas are often detected at an early stage due to distortion of the iris as a visible phenomenon. As the tumour enlarges the patient can experience blurred vision (ciliary body), decreased visual acuity, floaters, photopsia, and visual field defects. Exudative detachment of the retina can be seen, more rarely angle-
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closure glaucoma (13). Patients with susceptible lesions should be examined in a slit lamp followed by ultrasonography. Malignant tumours often present themselves as prominent, grey/brown tumours that have a circular/ oval shape. Orange pigmentation, tumour exudation and lower serous detachment can also be observed. Magnetic resonance imaging (MRI) and fluorescein angiography can be used to characterise the lesion. About 88% of UMs show low echogenicity on ultrasonography. Fluorescein angiography can detect risk features such as dye leakage and irregular vessels (4). Fine needle aspiration (FNAB) is valuable tool in stratifying lesions into malignant and benign categories, thus securing the initial diagnosis. Evaluation of metastatic spread is assessed by clinical examination, blood samples (including assessment of liver status), X-ray of the thorax and ultrasonography of the abdomen. Computed tomography (CT) scan and MRI can be used to specify unclear sonographic findings. Positron emission tomography (PET)/CT scan is not routinely performed (4, 14). Differential diagnosis of UM include choroidal nevus, intraocular metastases, congenital hypertrophy of the retinal pigment epithelium (RPE), haemorrhagic RPE detachment, choroidal haemangioma, age-related macular degeneration, RPE hyperplasia, among others (14, 15).
5.1.3 TNM classification and prognostic pathological parameters
Several factors have prognostic impact on UM, including histological parameters and extraocular extension. UM is staged according to the tumour, lymph nodes, metastasis (TNM) classification of malignant tumours (16). T describes the size of the original tumour and whether or not it has invaded nearby tissue. N tells of the involvement of lymph nodes, whereas M reports on the presence of metastatic disease. Due to a relative lack of lymphatic outflow from the eye, regional lymph node metastases are rare and can primarily be seen in cases of extra ocular extension of the tumour (17). Intratumoural lymphatics seen in some UM with extraocular extension are hypothesized to be recruited from conjunctival lymphatics (18). The presence of metastases and lymphatic spread impairs prognosis significantly (16). Cell type is assessed during routine histopathological examination and is an important prognostic indicator.
UMs are classified into three different subgroups according to cell morphology, namely Spindle, Epithelioid and Mixed tumours (Figure 2). Spindle celled UMs are characterised by an elongated nucleus and can be further divided into Spindle A and Spindle B cells. Spindle A nuclei lack nucleoli, while nucleoli is a characteristic feature of Spindle B nuclei. Epithelioid tumours resemble epithelium cells with eosinophilic cytoplasm, polygonal shape and prominent
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nucleoli. The definition of mixed tumours is elusive, a proposed definition is the presence of minimum 10% of Spindle or Epithelioid cells, most often the presence of >10% Epithelioid cells (14). Additional prognostic factors include tumour size, mitotic activity, status of extrascleral extension, mean diameter of the ten largest nucleoli, presence of mitotic figures, presence of lymphatic infiltrates and architecture of the microcirculation (4, 19). Important chromosomal abbreviations will be addressed more thoroughly in section “4.2 Genetic determinants in UM”.
Figure 2: Histopathology of Uveal melanoma: (A) Epithelioid cells and (B) Spindle cells.
5.1.4 Treatment
Small tumours (height < 2mm and diameter < 4mm) detected by routine examination can often be observed by regular fundus screening examinations. If the tumour shows signs of growth, intervention should be considered (4). For many years, enucleation was considered the sole method of treatment for larger UMs. Today, the advances in the field of radiotherapy have greatly increased the possibility of preserving the eye. The Norwegian Health Council recommends enucleation in cases where the tumour is large, has a substantial extrascleral outgrowth or encircles more than 180 degrees of the optic nerve. Transscleral local resection is a surgical option primarily for tumours localised anteriorly and is commonly followed by brachytherapy. This method is used for patients who are not candidates for radiation therapy, but are highly motivated to retain their eye. Plaque brachytherapy is the most widely used
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radiotherapy and offers a targeted delivery of radiation to the tumour. A small plate (plaque) containing seeds of ruthenium (Ru-106) or iodide (I-125) is attached episclerally of the lesion.
Another method, less commonly used, is proton beam therapy. This method can be used on larger tumours and on tumours localised closer to the optic disc/fovea. Proton therapy is currently not available in Norway, though selected patients can be treated abroad. Additional methods of treatment exist, these are rarely used as monotherapy/ curative treatment (20).
5.1.5 Management of metastatic disease
Despite advances in treatment of primary UM, the mortality rate has remained largely unchanged (4). Few treatment options exist for patients who develop metastatic disease. Local resection of liver metastases either by surgery or by stereotactic radiosurgery is reported to prolong survival. Unfortunately most patients present with diffuse involvement of the liver and will therefore not qualify for surgical resection. Isolated liver perfusion with melphalan, and hypothermia have been tested for patients with multiple liver metastases. Additional treatment strategies include chemoembolization or radioembolization with Yttrium 90 microspheres (4).
Over the last decade proto-oncogene B-Raf (BRAF), mitogen-activated protein kinase kinase (MEK) and checkpoint inhibitors have revolutionised the treatment of patients with cutaneous melanoma (CM). Most UM are not sensitive to BRAF inihibitors since they don’t harbour BRAF mutations (21). The use of MEK inhibitors is also questionable (22). Immunotherapy with immune checkpoint inhibitors (ICI) such as anti-cytotoxic T lymphocyte antigen (CTLA)- 4 (ipilimumab) or anti-programmed cell death protein (PD)-1 antibodies (pembrolizumab or nivolumab) has shown some effect. In a retrospective study, a partial response to first-line treatment was observed in 7% of patients treated with anti-PD-1 monotherapy and in 21% of those treated with combined anti-CTLA-4 plus anti-PD-1 therapy. The estimated one-year overall survival rate increased from 25.0% to 41.9% and the median overall survival improved from 7.8 months to 10.0 months (23).
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5.2 Genetic determinants in uveal melanoma
5.2.1 Cytogenetic features
Chromosomal aberrations are important determinants for metastatic spread. The loss of chromosome 3 is considered one of the most valuable prognostic markers. Monosomy 3 (M3) is associated with decreased survival and the presence of risk factors such as large tumour diameter, epithelioid cell type and extraocular extension (14, 24). Partial deletions of one copy of chromosome 3 and isodisomy also correlate with metastatic disease (25, 26). Gain of 8q (trisomy 8, isochromosome 8q and amplification of the c-Myc gene), in addition to M3, greatly impairs prognosis. The five-year disease specific mortality rate for M3 tumours is 40%. The co-existence with 8q gain increases the mortality rate to 66% (27). The loss of a part or all of chromosome 1 is another factor contributing to poor outcome and occurs more frequently in M3 tumours (24, 28). The loss of chromosome 6q is also associated with poor prognosis. In contradistinction to the aforementioned chromosomal changes, the gain of chromosome 6p is a predictor for better prognosis and is rarely seen together with M3 (estimated coexistence of 4%) (29). Abnormalities in the q-arm of chromosome 16 are relatively common in UM, though not associated with survival or other cytogenetic/ histopathological parameters (24).
A summary of the percentages of the main chromosomal aberrations in UM from various studies reviewed by Dogrusöz et al are shown in Table 1 (30).
Loss of 1p Monosomy 3 Gain of 6p Gain of 8q
Range 19-34% 25-65% 18-54% 37-63%
Table 1. Frequency of common chromosome alterations with evident prognostic significance. Summary of studies reviewed by Dogrusöz et al 2017
5.2.2 Molecular pathways and genomic alterations in UM
Despite their common embryological origin, the genetic characteristics of UM differs greatly from those seen in cutaneous melanoma. UM lacks mutations typically associated with CM and has a low mutational burden (31).
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Activation of the G protein subunit alpha 11/Q (Gα11/Q) pathway is important in early UM development and occurs in almost all primary UM via single amino acid substitution mutations in G protein subunit alpha Q (GNAQ) (57%) and G protein subunit alpha 11 (GNA11) (41%) (32-34). GNAQ and GNA11 encode two closely related G-alpha subunits that are components of G protein coupled receptor proteins (GPCR). GPCR receptors encompass numerous physiological functions and are critical in tissue homeostasis and cellular proliferation (35, 36).
Mutations in GNAQ and GNA11 are not sufficient for malignant transformation alone (33, 37).
Primary tumours that do not harbour mutations in GNAQ or GNA11 usually have mutations in the Gα11/Q pathway associated genes Cysteinyl leukotriene receptor 2 (CYSLTR2) and Phospholipase C β4 (PLCB4). CYSLTR2 encodes a G-protein coupled receptor and is constitutively activated in 4% of primary UM. PLBC4 activates signalling downstream by directly binding Gαq and is activated in 2.5%–4% of primary UM (31, 38, 39).
Mutated Gα proteins mediate the activation of the phospholipase C (PLC)/ protein kinase C (PKC) pathway and multiple downstream signalling pathways, including the rapidly accelerated fibrosarcoma (RAF)/MEK/ extracellular-signal-regulated kinase (ERK) pathway.
In addition to phosphoinositide 3-kinase (PI3K)/ AK strain transforming serine/threonine kinase (AKT)/ mammalian target of rapamycin (mTOR), and triple functional domain protein (Trio)/Ras homologous (Rho)/ Ras-related C3 botulinum toxin substrate (Rac)/yes-associated protein 1 (YAP1) pathway (40). Activation of the mitogen-activated protein kinase (MAPK) cascade is seen in up to 86% of primary UM (41, 42). The activation of PLC and subsequent cleavage of phosphatidylinositol diphosphate (PIP2) into inositol triphosphate (IP3) and diacylglycerol (DAG) results in activation of protein kinase C (PKC). PKC activates the MAPK pathway via targets including RAF, MEK and ERK, and results in transcription of genes involved in proliferation, differentiation and cell survival (Figure 3). The downstream activation of MEK has stimulated the testing of MEK inhibitors in the treatment of UM (22).
The PI3K/ AKT pathway is highly activated in many cancers and has been shown to promote proliferation and reduce apoptosis (Figure 3). AKT is a serine/ threonine kinase and is activated by phosphorylation. The phosphorylated AKT can further inactivate proteins involved in apoptosis and its expression correlates with poor prognosis in UM (43, 44). Phosphatase and tensin homolog (PTEN) acts as a tumour suppressor by negatively regulating the AKT/ protein kinase B (PKB) signalling pathway (45). Loss of heterozygosity of at least one PTEN marker has been demonstrated in 76% of primary UM, in addition loss of cytoplasmic PTEN expression
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is associated with cancer relapse (46). Downregulation of PTEN is suggested to be a late event in tumour progression due to its association with increased aneuploidy (29).
Gαq/11 signalling also promotes the activation of the Trio/Rho/Rac/YAP1 pathway (Figure 3).
YAP is hypothesized to promote the transcription of transcription factors associated with cell growth and viability and is a proposed therapeutic target (47).
In the majority of UMs, the p53 and retinoblastoma (Rb) pathways are functionally inhibited, although mutations in the TP53 and RB1 are rare (48, 49). Both pathways serve as tumour suppressors. The Rb protein prevents the cell from replicating damaged deoxyribonucleic acid (DNA) and can induce growth arrest in the G1 phase (50).
Figure 3: Oncogenic signalling pathways in UM. G-protein coupled receptors (GPCR) signal through the heterotrimeric proteins, Gα and Gβγ. Mutations in GNAQ or GNA11 lead to constitutive activation of Gα and downstream stimulation of the mitogen-activated protein kinase (MAPK) pathway via phospholipase C (PLCβ) and protein kinase C (PKC). The phosphotidylinositol-3 kinase (PI3K)/AKT/mTOR and the Yes-activated protein (YAP) pathways are also activated. Adapted from Park et al 2018 and Yang et al 2018.
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In addition to mutations in GNAQ, GNA11, PLCB4 and CYSTLR2, UM is characterised by mutations in three secondary driver genes; BRCA1 associated protein 1 (BAP1), Splicing factor 3B subunit 1 (SF3B1) and Eukaryotic translation initiation factor 1A, X-linked (EIF1AX).
BAP1
The strong association between M3 in UM and metastatic disease suggested that one or more tumour suppressor genes were located on chromosome 3. In 2010 Harbour et al discovered that BAP1, located at chromosome 3p21.1, was mutated in 47% of UM and in 85% of metastatic UM (51). BAP1 belongs to a specific group of proteases, called deubiquitinating enzymes (DUB). BAP1 is a catalytic component of the Polycomb repressive deubiquitinase complex (PR-DUB) that mediates deubiquitination of histone H2A monoubiquitinated at 'Lys-119' (H2AK119ub1), thus antagonising the activity of polycomb repressive complex 1 (PRC1) (52, 53). The exact role of BAP1 in gene regulation is still enigmatic. In healthy cells BAP1 removes ubiquitin from H2AK119 and thereby releases repression of transcription (54). Additionally, BAP1 binds and deubiquitinates the Transcriptional regulator host cell factor (HCF-1) (55, 56).
HCF-1 regulates gene expression by serving as a scaffold for chromatin remodelling complexes and by binding to several transcription factors (57, 58).
Enhancer of Zeste Homolog 2 (EZH2) is the enzymatically active core subunit of the Polycomb repressive core 2 complex (PRC2). PRC2 methylates the lysine residue at position 27 of histone 3 (H3K27), which facilitates chromatin compaction and gene silencing (59). EZH2 is opposed by the switch/sucrose non-fermentable (SWI/SNF) multiprotein complex. The SWI/SNF family of chromatin remodelling complexes serve to either enhance or suppress gene transcription through mobilization of nucleosomes (60). As cells differentiate, EZH2 activity is increasingly opposed by SWI/SNF, thus facilitating gene expression and terminal differentiation (61). EZH2 has the capacity to silence tumour suppressor genes and micro ribonucleic acid (microRNAs), but can also function as a gene activator (62, 63). The overexpression of EZH2 due to aberrant activation of EZH2 or loss-of-function mutations in the SWI/SNF complex is associated with cancer aggressiveness and advanced disease (64, 65). Loss of BAP1 function has previously been shown to increase EZH2, thus leading to EZH2 -dependent transformation (66). A recent study assessed whether EZH2 deletion could restore expression of BAP1-regulated genes.
Deletion of EZH2 in cells already depleted of BAP1 did not impair proliferation. Of the genes downregulated in BAP1 depleted cells, most of them remained silent in the EZH2/BAP1 double
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knockout model, the small set of genes upregulated in the double knockout model was also upregulated in BAP1 positive/ EZH2 negative cells. BAP1 promotes gene expression in a manner that is largely independent of an antagonism with the PRC2 complex (53).
A summary of BAP1 functions and interacting protein partners is presented in Figure 4 (67).
Figure 4: Summary of the functional roles of BAP1. BAP1 regulates the DNA damage repair pathway through interactions with BRCA1, BARD1 and RAD51. BAP1 interact with HCF1 in a number of processes involved in cell-cycle control and proliferation. BAP1 binds to ASXL to form the PR-DUB complex, responsible for regulation of chromatin through Histone H2A deubiquitination. BAP1 is associated in a number of regulated cell death pathways including apoptosis and ferroptosis. BAP1 is implicated in immune regulation. Courtesy of Louie et al 2020.
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SF3B1 encodes a core component of the ribonucleic acid (RNA) splicing machinery, the spliceosome processes precursor messenger RNA (mRNA) into mature transcripts and is located on chromosome 2q33. SF3B1 ensures correct splicing by retaining pre-mRNA to define the site for splicing, thus mutations in this gene can result in unique aberrant proteins but also in loss of expression (68, 69). Mutations in SF3B1 are detected in approximately 15% of UM cases (70). SF3B1 mutations are mainly restricted to tumours without M3, and are associated with late metastatic spread (34).
EIF1AX
The EIF1AX gene is located on chromosome Xp22 and approximately 17% of UMs harbour this mutation (31, 71, 72). EIF1AX has a role in initiating translation through a combination of stabilisation of the ribosome and recognition of target mRNA thus preparing mRNA for translation (73). EIF1AX encodes the eukaryotic translation factor 1A (eIF1A) that is essential in the transfer of methionyl initiator transfer ribonucleic acid (tRNA) to the small (40s) ribosomal unit (74). EIF1AX mutations are inversely associated with metastatic disease, most mutations are identified in tumours with disomy 3 (D3) (48%) and are rare in M3 tumours (3%) (71, 72).
5.2.3 Binary clustering of uveal melanomas
In 2004 Onken et al presented subclustering of UM into two distinct molecular classes based on gene expression profile. The division into Class 1 (1a low-grade tumours, 1b low-grade with metastatic potential) and Class 2 (high-grade tumours) was strongly correlated with cytological severity and survival (75). A significant association between genes expressed in Class 2 tumours and those expressed in primitive ectodermal and neural stem cells has also been demonstrated (51, 76). In 2010 the gene expression findings were commercialised as the DecisionDx-UM GEP test. The test migrates the initial findings into a 15-gene quantitative polymerase chain reaction (qPCR) assay with 12 discriminating genes and 3 control genes, and is claimed to be superior to assessment of M3 and clinicopathological prognostic factors for predicting metastasis (Table 2) (77-80).
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Table 2: Summary of the 15 genes tested in the DecisionDx-UM GEP test.
Gene symbol Gene name
Upregulated in Class 2 uveal melanoma
CDH1 E-cadherin
ECM1 Extracellular matrix protein
HTR2B 5-Hydroxytryptamine (serotonin) receptor 2B
RAB31 RAB31, member RAS oncogene family
Downregulated in Class 2 uveal melanoma
EIF1B Eukaryotic translation initiation factor 1 B
FXR1 Fragile X mental retardation, autosomal homolog 1
ID2 Inhibitor of DNA binding 2
LMCD1 LIM and cysteine-rich domain
LTA4A Leukotriene A4 hydrolase
MTUS1 Microtubule-associated tumour suppressor 1 ROBO1 Roundabout, axon guidance receptor, 1
SATB1 SATB homeobox 1
Control genes
MRPS21 Mitochondrial ribosomal protein S21
RBM23 RNA-binding motif protein 23
SP130 Sin3A-associated protein, 130kDa
The cost-benefit of the commercial test is a subject of debate. A prospective, 5-year multi- centre study has shown that Class 1A offers a 2% chance of the UM spreading over the next five years. Class 1B has a 21% chance of metastasis over five years, while Class 2, high risk UM, has 72% chance of metastasis within five years (81). Regardless of test results, long-term follow-up is of importance since metastatic disease is often seen within the first 10 years after diagnosis and can also be seen more than 25 years after treatment of the primary tumour (12).
It should also be noted that it is possible to receive both Class 1 and Class 2 test results in the setting of a non-melanoma malignancy, thus histopathology should be performed in addition to the DecisionDx-UM GEP test for correct diagnosis (82). The discovery of BAP1 loss/ mutation in aggressive UMs has raised the question whether immunohistochemistry (IHC) could be more cost effective since it can easily be implemented as a routine staining at Pathology Departments
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(83). Intratumoural heterogeneity and sampling errors are possible IHC drawbacks, though this has also been shown for the DecisionDx-UM GEP test (84).
A modified version of the DecisionDx-UM GEP test that includes preferentially expressed antigen I melanoma (PRAME)) is also commercially available. PRAME has been shown to be an independent biomarker for metastasis in UM. PRAME positivity is associated with an increased risk of metastasis in Class 1 tumours and a shorter time to metastasis in Class 2 tumours. If a tumour is negative for PRAME, the prognosis indicated by the DecisionDx-UM Class is not expected to be altered (85, 86).
More recently, Class 1 and Class 2 tumours have been further divided into the subcategories A- D (87). The subdivision was based on data from the Cancer Genome Atlas, where primary tumour material from 80 patients with UM have been analysed for histologic features, chromosome copy number, genetic mutations, expression of RNA, proteins, DNA methylation status, in addition to factors such as biochemical pathways and immune markers (39).
A summary of driver and secondary genetic alterations in UM development and progression is shown in Figure 5, included in the figure is serine and arginine rich splicing factor 2 (SRSF2) associated with Class 1b tumours (40).
Figure 5: Acquisition of driver and secondary genetic alterations drive uveal melanoma (UM) development and progression. The sequential acquisition of genetic changes (highlighted within the vertical arrows) leads to distinct genetic profiles that reflect the risk of UM metastases. Courtesy of Park et al 2018.
23 5.2.4 Genetic alterations in metastatic UM
The emergence of metastatic disease in UM can be seen months to decades after primary surgery and the latency period could reflect the time needed to acquire distinct oncogenic alterations (88).
Kiilgaard et al performed DNA sequencing of 35 primary UM and matched metastases (89). In contradistinction to several other cancers, the metastases of UM tend to have more oncogenic mutations than primary UM (90). The study showed that copy number (CN) changes of 6p, 1q and gains of 8q were enriched in metastases. This was in concordance with previous publications detecting chromosome 3 monosomy (73%), 8q gain (89%), 6q loss (64%), 1p loss (47%), 8p loss (45%), 1q gain (35%), and 16q loss (32%) in liver metastases (91, 92). The amplitude of 8q tended to increase from primary tumour to metastases. As expected in metastatic disease, the number of UM with SF3B1 or EIF1AX mutations was low (n=7). These cases showed additional oncogenic alterations in e.g. CDKN2a and PTEN. In one EIF1AX mutant case a part of the tumour had acquired a deletion of chromosome 3. Mutations in chromatin remodelling factors were also observed, including mutations in polybromo 1 (PBRM1) and EZH2.
5.3 Epigenetics
Epigenetics is a rapidly developing field in clinical medicine and biomedical research, and is considered to be one of the hallmarks of cancer (93-95). An epigenetic trait is the constantly- heritable phenotype resulting from changes in a chromosome without alterations in the DNA sequence (96). At least three types of epigenetic modifications regulate chromatin conformation: DNA methylation, histone modifications, and non-coding RNAs. Histone modifications are posttranslational modifications of histone proteins which includes methylation, acetylation, citrullination, SUMOylation, phosphorylation, ADP-ribosylation and ubiquitination (97). Histone modifications can activate or silence transcription by controlling the accessibility of DNA to the transcriptional machinery and by protein interactions (98, 99).
Non-coding RNAs (ncRNAs) function to regulate gene expression at the transcriptional and post-transcriptional level and can be divided into two main groups, namely short ncRNAs (<30 nucleotides) and long ncRNAs (>200 nucleotides). Micro RNAs (small (≈22 nucleotides), single stranded, non-coding RNAs) are among the most studies ncRNAs and can repress gene expression by binding to complementary sequences of mRNA thereby preventing their
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translation. MiRNAs can drive tumorigenesis by overexpression of oncogenic miRNAs (oncomirs) or by loss of tumour suppressor miRNAs (100, 101).
Differential DNA methylation in UM was the subject of interest in paper II and the process of DNA methylation will therefore be highlighted in section 5.3.1.
5.3.1 DNA methylation
In mammalian cells, DNA methylation occurs almost exclusively at the C-5 position of cytosine (5mC) in cytosine-phosphate-guanine (CpG) nucleotides. The majority of CpG nucleotides in the genome are methylated and most of the methylated CpGs are located in regions with low density of CpGs. Of the approximately 28 million CpG sites present in the human genome, 60- 80% of the cytosines are methylated as 5mC (102). Regions of the genome that are enriched in CpG repeats are referred to as CpG islands (CGIs). A CGI is defined as a region of DNA > 200 base pairs with a GC content ≥50%, and the ratio of observed/expected CpG >0.6 (103). CGIs are present in or near approximately 40% of gene promoters (104). Although the bulk of genome is methylated at its CpGs, CGIs are mostly unmethylated in somatic cells (105). In general, hypermethylation of promoters is associated with gene silencing, while methylation of gene bodies is often a permissive mark (106, 107).
The methylation of cytosines is mediated by a class of enzymes called DNA methyltransferases (DNMTs) and involves the transfer of a methyl group from S-adenosyl-methionine (SAM).
Five members of the DNMT family have been identified, but only three possess an inherent enzymatic activity (DNMT1, DNMT3a, DNMT3b). DNMT1 is a maintenance methyltransferase, ensuring the methylation status of each CpG during replication (108, 109).
DNMT3a and DNMT3b are essential for de novo methylation and mammalian development (109). DNA methylation is assumed to interfere with transcription by either physiologically impede the binding of transcriptional proteins to the gene or by recruiting methyl-CpG-binding domain proteins (MBD) to methylated DNA. MBD proteins can further recruit proteins involved in chromatin remodelling and induce conformational changes and silencing (110, 111).
In the absence of functional DNA methylation maintenance machinery, 5mC can be lost during successive rounds of replication, thus leading to passive DNA demethylation. By contrast, active DNA methylation refers to an enzymatic process that removes or modifies the methyl group from 5mC (112). Active demethylation by oxidation is achieved by Ten-eleven
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translocation (TET) -enzymes (TET1, TET2, TET3). These enzymes convert unmodified 5mC to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC), followed by excision of 5fC or 5caC mediated by thymine DNA glycosylase (TDG) coupled with base excision repair (BER) (113). Another proposed mechanism for demethylation of 5mC involves the deamination of 5mC and 5hmC by the deaminase enzymes activation-induced cytidine deaminase (AID)/ apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like APOBEC (114). An illustration of DNA methylation addition, maintenance and removal is presented in Figure 6 (115).
Figure 6: DNA methylation predominantly occurs at the fifth carbon atom of cytosine bases. Its deposition is catalysed by the de novo DNMTs DNMT3A and DNMT3B. Introduced methylation patterns are preserved by the maintenance DNMT DNMT1 during replication. Passive DNA demethylation is considered to be achieved across cell division in the absence of DNMT1 maintenance activity. Active removal includes the mammalian TET1–3 proteins that are capable of converting 5- methylcytosine to its oxidised derivative 5-hydroxymethylcytosine (5hmC) and further to 5- formylcytosine and 5-carboxylcytosine (not indicated here). Courtesy of Ambrosi et al 2017.
Cancer cells display an aberrant methylation pattern recognised by global hypomethylation and hypermethylation of promoter associated CpGs (116, 117). The global DNA hypomethylation in cancer is mainly due to hypomethylation of highly repetitive DNA sequences e.g. short interspersed nucleotide elements and long interspersed nucleotide elements (118-120). The dense methylation of these regions as seen in normal tissue presumably maintains genomic integrity by preventing translocations, genomic disruptions and genomic instability (117, 121- 124). The aberrant hypermethylation of promoters seen in cancer, is often associated with the
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silencing of tumour suppressor genes such as BRCA1 and Von Hippel-Lindau tumour suppressor (VHL) (106, 125, 126). Rarely, promoter methylation can also serve as a permissive mark (127). The exact role of promoter methylation in gene silencing is a subject of discussion and could be a late event in gene silencing, secondary to nucleosome positioning (128-130).
Even though the understanding of promoter methylation in gene silencing is in its beginning, the link between cancer and methylation is undeniable. The lifetime risk of cancer is correlated to the degree of abnormal methylation changes that occur during the ageing of normal tissue.
Tissue where the cells have a relatively high degree of abnormal methylation (e.g. colon) has a higher propensity for developing cancer than cells characterised by a lower degree of aberrant methylation (131).
Despite controversy over the role of promoter methylation in gene silencing, DNMT inhibitors have shown promise in the treatment of various cancers, especially haematological cancers (132-134). The differential effect of DNMT inhibitors in diverse cancer could be due to their heterogeneous methylation pattern (135). It should be noted that DNMT inhibitors might exert their effect by other mechanisms than promoter demethylation (107, 136). Intriguingly a DNMT inhibitor prodrug has been shown to have the ability to up-regulate HLA class 1 antigens, thus indicating a potential in increasing immunogenicity and immune recognition of neoplastic cells (137).
Relatively few studies have investigated the methylome of UM, though hypermethylation has been shown in areas associated with promoters for genes regulating the cell cycle, and extracellular matrix degradation, e.g. p16INK4a, RASSF1a, RASEF, Embryonal fyn-associated substrate (EFS) and Metalloproteinase inhibitor 3 (TIMP3) (138-142).
The global methylation profile of UM has been shown to coincide with clustering into Class 1 and Class 2 tumours (39, 143). Robertson et al showed that EIF1AX-mutant tumours were restricted to DNA methylation cluster 1, while UM in DNA methylation clusters 2 and 3 were highly enriched with tumours harbouring SF3B1/SRFR2 mutations. Thus, D3 UM with EIF1AX versus SF3B1/SRFR2 mutations possess distinct DNA methylation patterns. Monosomy 3 (M3)/BAP1-aberrant UM tumours showed a single global DNA methylation profile (39).
Interestingly partial deletion of chromosome 3 is associated with low- risk Class 1 UMs (144), thus raising the question why complete loss of chromosome 3 is required for Class 2 GEP. This was further investigated by Harbour et al who showed that the most significant and densely clustered hypermethylated/ downregulated gene loci in Class 2 UMs were located on chromosome 3, which contained many of the axon guidance cues, neural crest specification,
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and melanocyte differentiation genes (e.g., Roundabout homolog 1 (ROBO1), Plexin B1 (PLXNB1), Semaphorin-3B (SEMA3B), Cell adhesion molecule L1 like (CHL1), Special AT- rich sequence-binding protein-1 (SATB1), Microphthalmia-associated transcription factor MITF, Dishevelled Segment Polarity Protein 3 (DVL3), and Rapidly accelerated fibrosarcoma -1 Proto-Oncogene, Serine/Threonine Kinase (RAF1). Since these genes undergo repressive methylation changes on the sole remaining copy of chromosome 3, it could explain why the other copy of chromosome 3 must be lost to acquire the metastasising Class 2 UM phenotype.
Additionally a novel hypermethylated site within the BAP1 locus was found in all Class 2 tumours, suggesting that BAP1 itself is epigenetically regulated (145).
Characterisation of the methylome provides mechanistic insight into the development and progression of UM and could lay the foundation for the development of new therapeutics.
Methylation profiles can also serve as diagnostic and prognostic markers in addition to predicting responsiveness to therapy and monitoring of response (146-152).
5.3.1.1 DNA hydroxymethylation
The conversion of 5mC to 5hmC by TET enzymes has gained considerable attention as 5hmC has been shown to be a relatively stable epigenetic mark whose role in transcription regulation is linked to its genomic location (153). The 5hmC levels vary between different cell types and tissues and are highest in neurons, while cancer cells have lower levels compared to corresponding normal tissue (154, 155). Once cancer is formed, a lower level of 5-hmC correlates with poor prognosis (156, 157). 5hmC has a greater relative abundance in gene bodies compared to gene promoters, where 5hmC modified CpGs are generally depleted. An enrichment of 5hmC CpGs over enhancer elements and some transcriptional start sites is associated with silenced genes, while gene body methylation is often associated with active genes (158-162).
5.4 In vitro and in vivo preclinical models for studying UM
In vitro and in vivo models play a pivotal role in basic and translational cancer research and are important tools to investigate the pathogenesis of metastatic UMs and for drug testing.
Two-dimensional (2D) monolayer cultures of primary tumour cells and cell lines are indispensable in UM research and allow for expansion of cells and drug testing under direct
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visualisation. At the same time, these cultures represent non-physiological conditions that might not be representative for cancer cells residing in the complex microenvironment of primary tumours and metastatic niches.
Three-dimensional (3D) cultures are hypothesised to recapitulate in vivo growth including cell connectivity, polarity, tissue architecture and gene expression (163). That said, 3D cell culture models are often more time consuming, difficult to implement in standard workflows and often pose a challenge in imaging and quantitative analyses (164).
5.4.1. Three-dimensional in vitro models
Broadly, 3D cell cultures are classified as Scaffold-based (cells grown in presence of a support) and Scaffold-free techniques.
5.4.1.1 Scaffold based techniques
Scaffolds used for 3D cell cultures range from extra cellular matrix (ECM)-like matrices to simple mechanical structures and can be further divided into hydrogels and solid-state scaffolds.
Hydrogels are water swollen polymeric material and include natural hydrogels (e.g. agarose, laminin, collagen, hyaluronic acid) and synthetic hydrogels. Matrigel is an example of a natural hydrogel and is a solubilised basement membrane preparation extracted from the Engelbreth- Holm-Swarm mouse sarcoma and provides ECM protein such as laminin, collagen IV, proteoglycans and a number of growth factors (165). Solid state scaffolds have the ability to organise positioning of cells in a reproducible and controllable manner (166).
5.4.1.2 Scaffold-free techniques
Scaffold-free 3D cultures facilitate the formation of spheroids (multicellular aggregates) (167).
The formation of spheroids relies on either forced or self-assembled clustering of cells.
Hanging drop cultures involves the culturing of cells in a drop of media suspended in the lid of a cell culture dish, meaning that the drop has to be small enough to adhere to the lid under manipulation. Aggregation can also be promoted using plates with low attachment coating (low adhesion plates), these plates have a higher volume capacity than the hanging drop method and often result in the formation of one spheroid per well. An additional technique is magnetic
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levitation where cells are preloaded with magnetic nanoparticles and further aggregated using an externally applied magnetic field. Spheroids can also be generated with the aid of bioreactors e.g. spinner flasks and rotational bioreactors. Bioreactors provides greater reproducibility and can produce a larger number of uniform spheroids. Another way to introduce flow to cell culture systems is through the use of microfluidic devices. These devices contain micro-channels, thus allowing for continuous delivery of nutrients and the creation of gradient concentrations of biochemical signals (168, 169).
5.4.2 In vitro assays for studying the metastatic process of UM
The dismal outcome of metastatic UM urges the need for validated models to study the mechanisms controlling metastasis. The sequential steps of metastasis include the degradation of ECM, intravasation into blood vessels, circulation within the bloodstream, attachment to the endothelium of a target organ and the extravasation into connective tissue before proliferation (170). The complex process of metastasis renders a uniform model unlikely since a thorough understanding of every step is needed. Several in vitro models for studying metastasis have been developed, each with its strengths and limitations.
Migratory and invasive capacity are prerequisite skills for metastatic spread. Boyden chamber assay and its modifications can be used to study invasion, chemotaxis (migration towards a chemical concentration gradient) and haptotaxis (ECM protein gradient) of tumour cells. The standard Boyden chamber assay involves the seeding of cancer cells on top of a transwell membrane suspended over a larger well which contain medium/ chemoattractants. Cells are allowed to migrate through the porous membrane before migratory cells are stained and counted, modifications involve the addition of e.g. Matrigel on top of the membranes and addition of feeder layers. Migratory cells can be detected and quantifies by both colorimetric and fluorometric methods (171).
A simple and well-developed method to assess cell migration is the scratch assay. This method introduces a “scratch” in a monolayer cell culture and images are captured at the beginning and at regular intervals. Images are then compared to quantify the migration rate of the cells. The Ring assay uses the same concept as the scratch assay, cells are allowed to grow to confluency before a central ring is removed, allowing cells to migrate into this area. Cell migration can also be evaluated by microcarrier bead assay, where cells are grown on microcarriers before being
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transferred to plastic wells, migration to the plastic wells is assessed after removal of the beads (171, 172).
The use of the previous mentioned microfluidic devices is emerging as these small chambers enables control over local gradients, fluid flow, tissue mechanics, and composition of the local environment. Additionally, these chambers can be made optically accessible for live observation. Microfluidic devices could represent a valuable substitute for animal models in preclinical trials (173).
5.4.3 In vivo assays for studying UM
Great advancements in medical research have been attributed to the use of animal models and these models are still valuable tools in cancer research. The use of animal models raises several ethical questions and the three R`s (Replacement, Reduction and Refinement) should always be taken into consideration (174). In cancer research, a well-designed animal model can provide insight into basic pathobiology and the process of metastatic spread. Testing of novel therapeutics also rely in these models, as animal models represent a bridge between in vitro research and clinical trials.
Animal models can be divided into spontaneous models, transgenic models and induced models. The relatively low incidence of UM, even in animals, limits the use of spontaneous models.
The genetic engineering of transgenic animal models allows oncogenes to be constitutively or conditionally expressed and tumour suppressor genes to be silenced (175). In cutaneous melanoma, numerous transgenic mouse models have been successfully established. Attempts in developing transgenic models in UM have been undertaken, including the development of a GNAQ mutant mouse strain, unfortunately these models have failed to develop liver metastases, thus no transgenic models are currently available for UM (176, 177).
Induced animal models involves the artificial introduction of disease by radiation, chemical agents, viruses, cells or tissues. Several induced animal models exist, including intraocular, intrasplenic, intravenous and intrahepatic injections of tumour cells in addition to patient derived xenografts. It should be noted that the ability to grow metastases is tumour dependent, and that these models rely on immunocompromised animals (171, 176, 177).
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6. Aims of the thesis
The overall aim of the thesis is to shed light upon underlying mechanisms in the development and progression of UM, thus unravelling potential treatment strategies and improve prognostic assessment.
More specifically:
1) In the first paper, our aim was to compare the differential gene expression of multicellular tumour spheroids (MCTS) of UM to primary tumour tissue and adherent cultures, with a special emphasis on unravelling the pathways and survival mechanisms pathognomonic for disseminated and circulating cancer cells.
2) In the second paper, we sought to delineate biologically relevant groups and genes in FFPE derived UM specimens by correlating histopathological data and survival data of the patients with methylation profiles and gene expression.
3) The aim of the third paper was to investigate the differential expression of Cx43 in primary UM biopsies and cultures vs healthy choroidal tissue and choroidal melanocytes and explore potential regulatory mechanisms.
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7. Methods and methodological considerations
All experiments were performed in accordance with the Declaration of Helsinki. Both tissue harvesting and the use of archived paraffin embedded tissue blocks were approved by the local Committees for Medical Research Ethics. Fresh tissue samples were obtained after informed written consent.
7.1 In vitro cultivation
Various procedures for preparation of single cell suspensions from tumour tissue exist. The rate of success is determined by dissection procedure, tissue quality, method of separation (enzymatic or filtration). The use of enzymatic digestion is dependent on enzyme used, concentration, temperature, and length of incubation. The most widely enzymes used include trypsin, collagenase, dispase, hyaluronidase, papain and elastase. Other commercially available solutions include Accutase (Innovative Cell Technologies, Inc., San Diego, US) and TrypLE (Thermo Fisher Scientific, Waltham, US), enzymes that allegedly cause less damage than trypsin. Our method of choice was based on personal experience and published literature. Other groups have had success with non-enzymatic separation of tumour tissue (filter, cloth, mincing) due to small sample size we preferred an enzymatic approach (178). In our experience dissociation in 0.25% Trypsin digestion often resulted in an overgrowth of fibroblasts or cell death in adherent cultures (179). In the first paper, UM tissue was obtained from patients undergoing enucleation of the eye. After surgery, the eye was transferred to a 0.9% NaCl and transported to the Pathology Department where an Ophthalmological Pathologist excised a small portion of the tumour for research purposes. The tissue was minced into small pieces in a mixture of 1mg/ml of collagenase I and IV, before incubation for 1 h at 37C. The pellet was resuspended in RPMI 1640, 10% FBS, 0.5% Penicillin/Streptomycin and 0.25% Amphotericin B. Gentamycin 75µg/ml was added to ensure removal of fibroblasts from the cell culture (180).
This protocol was kindly provided by Tina Maria Ludowika Jehs from the University of Copenhagen and was originally intended for isolation of uveal melanocytes. In our experience, the protocol results in a homogenous cell culture viable for 1-3 passages if the quality of the starting material is satisfactory (data not shown). In addition we tested a neuronal dissociation kit from as described by Tura et al, our results indicated increased cell yield using this method
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(181). Unfortunately the miniscule samples we obtained for research purpose did not allow for parallel testing of the isolation protocols.
RPMI 1640 was chosen over DMEM/F12, as the latter was seemingly more favourable for fibroblasts (182). Alpha-MEM is proposed to be superior to RPMI1640, implementation of this media to our protocol could thus optimise culture conditions further (183).
For establishment of spheroid cultures, cells were grown for 7 days as adherent cultures before trypsinisation in a 0.25% solution under careful supervision. The cells were resuspended in hESC +MEF, 0.5% Penicillin/Streptomycin and 0.25% Amphotericin B and transferred to ultra-low attachment plates (Corning, Sigma-Aldrich, St. Louis, Missouri, United States). This media was chosen based on studies on skin melanomas (184). The use of cone shaped wells and the fact that the UM cells retained their pigmentation enabled us to change ¼ of the media every second/third day without disturbing the cells.
In the third paper we used a modified protocol developed by co-author Kirankumar Katta. This protocol implemented elements from isolation protocols of choroidal melanocytes. Briefly, the samples were treated with Dispase II, filtered and cultured in Ham`s F12 with 10% FBS and antibiotics.
7.2 Immunohistochemistry
IHC is the demonstration of antigens in tissue sections by the use of labelled antibodies as specific reagents through antigen-antibody interactions that are visualised by a marker. IHC does not only allow visualisation of proteins, but also allows the user to determine the subcellular location and/or co-localisation of them (185). Successful detection of antigens by IHC depends on a variety of factors, starting with tissue sample and fixation. Tissue should be rapidly preserved to avoid the breakdown of cellular proteins and tissue architecture. Formalin is the most commonly used fixative and was used in all experiments included in this thesis.
Fixation time should be standardised for all tissues. Prolonged fixation in formalin can result in excessive cross-linking, thus making the antigen unrecognisable for the antibody. Fortunately most antigens can be demasked if a proper method of antigen retrieval (AR) is used (186).
Under-fixation of samples is considered a more serious problem than over-fixation, since the core of the sample will only be fixated by alcohol before immersion in paraffin, thereby creating a heterogeneous fixation throughout the sample (185). Spheroid derived cells from paper I were fixated at 4oC overnight. Formalin-fixed, paraffin-embedded (FFPE) tissue from the diagnostic
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biobank was processed according to their standardised protocols. After fixation in formalin, the tissue is dehydrated in a series of xylene and alcohol dehydration before embedding in paraffin.
The effect of long-term storage of FFPE tissue is debatable, FFPE blocks can be stored for >25 years if stored at a cool place, FFPE slides should be limited for 7 days, though loss o f antigenicity is also suggested to be antigen dependent (187). Paper II and III included the use of archived FFPE tissue for IHC. In the context of UM research, assessment of BAP1 status is advisable. Staining of BAP1 was performed. Unfortunately the staining was inconclusive for several of the samples due to negative innate control (data not shown in publications), reduced BAP1 antigenicity in old FFPE tissue has also been encountered by other groups (188).
The tissue used in our experiments was sectioned at 3.5-4μm, since thicker sections can lead to difficulties in the interpretation of the staining due to multi–layering of the cells. After sectioning, the tissue was dried before further processing. This should be done at temperatures less than 60 degrees to avoid loss of antigenicity. Our standard protocol included drying for 1h at 59 degrees and overnight at 38 degrees. To prevent detachment of sections during AR, super frost slides were used (Thermo Fisher Scientific).
AR is an essential step in order to reverse the changes induced by fixation. The choice of AR depends on the targeted antigen and the type of antibody (185). Length of treatment, temperature, pH, and chemical composition of the AR solution are major factors that influence the effect of AR. To subgroups of AR exist, namely heat induced epitope retrieval (HIER) and proteolytic induced epitope retrieval (PIER). HIER includes water bath (PT-link), pressure cooker heating and microwave heating. PIER consists of various methods of enzymatic degradation. Our experiments were conducted using PT-link HIER. We also tested microwave heating and enzymatic digestion with trypsin, in our experience these were inferior to the PT- link for AR for the chosen epitopes. After AR the sections were treated with a blocking solution.
The time period of blocking is critical since prolonged treatment can result in masking of antigen and too short incubation time can result in non-specific binding of the secondary antibody. Blocking solutions include commercial blocking buffers, milk and serum. The choice of serum should be the same species as the secondary antibody is generated in (189). Our samples showed considerable less fluorescent background staining if the samples were treated with goat or donkey serum compared to bovine serum albumin (BSA) or milk. For fluorescent staining we used a concentration of 10% serum in phosphate-buffered saline (PBS). The dilution of primary antibodies was chosen based on testing of multiple dilutions, dilutions used in previous publications and recommended dilution from the supplier. Monoclonal antibodies