Cover Page The handle

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Cover Page

The handle http://hdl.handle.net/1887/61625 holds various files of this Leiden University dissertation.

Author: Doğrusöz, M.

Title: Genetic prognostication in uveal melanoma Issue Date: 2018-04-17

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Genetic Prognostication in Uveal Melanoma

Mehmet Doğrusöz

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Genetic Prognostication in Uveal Melanoma ISBN/EAN: 978-94-6299-945-9

Printing: Ridderprint BV Author: Mehmet Doğrusöz Thesis layout: Mehmet Doğrusöz Cover design: Mehmet Doğrusöz

The research described in this thesis was financially supported by Horizon 2020 UM CURE grant# 667787 (European Union), The Eye Cancer Network (New York, NY, United States of America), and Stichting Blinden-Penning (Amsterdam, The Netherlands).

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Genetic Prognostication in Uveal Melanoma

Proefschrift

ter verkrijging van

de graad van Doctor aan de Universiteit Leiden, op gezag van de Rector Magnificus prof.mr. C.J.J.M. Stolker,

volgens besluit van het College voor Promoties te verdedigen op dinsdag 17 april 2018

klokke 16:15 uur

door

Mehmet Doğrusöz

Geboren te Adana, Turkije in 1988

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Promotoren Prof.dr. M.J. Jager Prof.dr. G.P.M. Luyten

Leden Promotiecommissie

Prof.dr. J.F. Kiilgaard Universiteit van Kopenhagen (Denemarken) Dr. E. Kiliç Erasmus Universiteit Rotterdam

Prof.dr. C.J. van Asperen

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Chapter 1 General Introduction and Thesis Outline

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INTRODUCTION TO UVEAL MELANOMA Epidemiologic characteristics

Uveal melanoma (UM) comprises approximately 3-5% of all types of melanoma, most of which occur in the skin.^1-4 UM is an ocular tumor and develops from melanocytes residing in the iris, ciliary body, or choroid. The choroid is the most common (≈90%) site of origin, followed by the ciliary body (5-8%), and iris (3-5%).⁵ Both eyes are affected in equal numbers, and a bilateral occurrence of UM is very rare.⁶ UM has a mean annual age-adjusted incidence of 5.1 per million in Western countries and is the most frequently occurring primary intraocular malignancy in adults.⁴ Up to 80% of patients are over 50 years of age, with a mean age at diagnosis of 61.⁴ Albeit several studies reported no gender difference in incidence,^{7, 8} a recent study involving over 7,500 UM cases reported a slight but significantly different male-to-female ratio of 1.1:1.⁴ There is a racial and ethnic variation in its incidence. The incidence is 6 per million per year in whites, 1.67 in Hispanics, 0.38 in Asians, and 0.31 in blacks.⁹ Northern Europe has a higher incidence of UM than Southern Europe,¹⁰ with an annual incidence of more than 8 per million in Denmark and Norway and less than 2 per million in Spain and Southern Italy.¹¹ The incidence of UM in the United States has remained stable over the last decades,¹² while in Sweden, an annual relative decrease of 1% in males and 0.7% in females occurred between 1960 and 1998.¹³

Risk factors

Many host and environmental parameters have been evaluated as possible predisposing factors for UM development. Caucasians with a light iris color and a fair skin that burns easily after sun exposure have been shown to have a higher risk of developing UM than persons with a dark skin and eye color.¹⁴ This

association corresponds with the aforementioned race-dependent disparity in UM incidence, and the south-to-north increasing incidence in Europe: Northern Europeans have on average lighter eye pigmentation than Southern Europeans.¹⁵ Besides skin and eye color, other factors have been identified as host

susceptibility factors for developing UM. Oculo(dermal) melanocytosis (nevus of Ota) is a hyperpigmentation of the uvea, sclera, and episclera as well as of the periocular skin.¹⁶ This condition affects 0.04% of the white population, while the prevalence in UM patients is 1.2-3%, which makes this condition 30 to 75 times more prevalent in UM patients than in the general white population.^16-18 Individuals with oculo(dermal) melanocytosis have a 1 in 400 lifetime risk of

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developing UM, while the lifetime risk for UM in the general population is 1 in 13,000.¹⁹ Another risk factor is the presence of a choroidal nevus. Choroidal nevi are quite frequent in Caucasians, with a prevalence ranging from 5% to 8%.²⁰ The risk of malignant transformation of a choroidal nevus has been estimated to be 1 in 4,300 to 1 in 8,845 per year.^20-22 The risk may depend on the size of the nevus.

The rate of transformation of giant choroidal nevi (≥ 10mm diameter) into melanoma has been reported to be 18% at 10-year follow-up.²³

Besides choroidal nevi, the presence of common/atypical cutaneous nevi, familial atypical multiple melanoma mole (FAMMM) syndrome, and cutaneous freckles is associated with a higher risk of developing UM.^24-26

Recently, germline mutations in the BAP1 (BRCA1-associated protein-1) gene were found to confer a higher risk of UM as well as other malignancies, such as cutaneous melanoma, mesothelioma, meningioma, renal cell carcinoma, and lung adenocarcinoma.^{27, 28} We do not yet know how often Dutch UM patients carry this germline mutation. A study in Finland showed that 2% of their UM patients were affected.²⁹

In contrast to cutaneous melanoma, there is no conclusive evidence for an association between ultraviolet light exposure and the risk to develop UM. While a meta-analysis regarding the relation between ultraviolet radiation and the risk to develop UM yielded contradictive results, it identified welding as a possible risk factor.³⁰ This may be due to occupational exposure to artificial ultraviolet light in welders; however, welding arcs also emit blue light, which has recently been associated with the risk of developing UM.³¹ Dietary habits, smoking and alcohol consumption do not seem to affect the incidence of UM.²⁶

Presentation and Diagnosis

In a retrospective review of 2384 UM patients in an ocular oncology center, the most common symptom patients presented with was blurred vision (38%), followed by photopsia (9%), floaters (7%), visual field loss (6%), a visible tumor (3%), pain (2%), and metamorphopsia (2%). Approximately one-third of patients were asymptomatic on referral.³²

The symptoms caused by UM depend on the location and size of the tumor.

Patients with an iris melanoma are usually asymptomatic, and differentiation between a nevus and a melanoma is often difficult. The tumor may be noticed as a dark spot on the iris or have caused a distortion of the pupil. Most iris

melanomas (45%) are located in the inferior quadrant. Approximately 80% of

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cases are pigmented.³³ In a series of 200 patients with suspect iris lesions, 24%

cases were confirmed to be UM, while most other patients were diagnosed with iris cysts (38%) and iris nevi (31%).³⁴ The criteria for the clinical diagnosis of melanoma were a diameter larger than 3 mm and thickness over 1 mm, replacement of the stroma of the iris, and the presence of at least 3 of the following features: growth, secondary glaucoma/cataract, prominent vascularity, or ectopion irides.³⁴

In contrast to iris melanoma, a UM located in the ciliary body is not easily visible on ophthalmic examination and may be missed in the absence of symptoms.

Especially small ciliary body melanomas are hidden behind the iris and may not cause symptoms. Signs that may raise the suspicion of a ciliary body melanoma are dilated episcleral vessels (sentinel vessels), extrascleral extension, cataract when the tumor touches the lens, and raised intraocular pressure if the tumor grows circumferentially. The majority of ciliary body melanomas, however, grows in a dome-shaped configuration and may be visible on dilated fundus

examination.³⁵ Choroidal melanomas may be easily detectable, especially if they are located centrally, and may cause symptoms of e.g. vision loss and

metamorphopsia when located close to the macula. Peripheral choroidal melanomas may present with a visual field defect or with photopsia. Most choroidal melanomas (77%) grow in a dome-shaped configuration and are completely or partially pigmented (85%).^{36, 37} Although large and medium-sized tumors can often be accurately diagnosed by fundoscopy, the diagnosis of small melanomas can be more challenging due to their resemblance to nevi. The mnemonic “To Find Small Ocular Melanoma Using Helpful Hints Daily” (TFSOM- UHHD) which sums up risk factors for the transformation of a nevus into

melanoma has been proposed as a useful tool to identify small melanomas or to determine the follow-up schedule of melanocytic lesions.³⁸ A tumor thickness of >2 mm, the presence of subretinal fluid, visual symptoms, or orange pigment, a margin within 3 mm of the optic disc, ultrasonographic hollowness, absence of a halo (a circular band of unpigmented area surrounding a pigmented nevus) and lack of drusen have been identified as the relevant risk factors that predict growth of choroidal nevi into melanoma.³⁸ As evident from the inclusion of

ultrasonography (USG) in this mnemonic, the clinical diagnosis of UM can be supported or confirmed using various ancillary examinations. USG, especially B- mode ultrasound, is the most often used auxiliary method in the diagnosis of UM.

On B-mode USG, the tumor may appear as a dome- or mushroom-shaped hyper-

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echoic mass with a hollow appearance, due to a lower reflectivity than the surrounding choroidal tissue.³⁹ USG may be especially helpful in the diagnosis of UM in the presence of dense cataract or vitreous hemorrhage. Moreover, it can be used to measure tumor elevation which aids in treatment planning and the subsequent evaluation of the effect of eye-preserving treatments.^{35, 40}

Fluorescein angiography (FA) is valuable in confirming the presence of orange pigment and subretinal fluid, which are helpful in identifying small melanomas.⁴¹ FA may also reveal the presence of e.g. hyperautofluorescent drusen, which are indicative of a nevus.⁴²

The accuracy of diagnosing a UM has been estimated to be over 99% when the diagnosis was based on fundoscopy, USG, and FA.⁴³ However, a more recent study in 2,384 patients diagnosed with UM in an ocular oncology center reported that 23% of UMs had initially been missed.³²

Computer tomography (CT) and magnetic resonance imaging (MRI) are less commonly utilized in the diagnosis of UM and may be of use when

ultrasonography is unable to visualize the lesion in patients with media opacities such as dense cataract and vitreous hemorrhages.⁴¹ MRI may be especially helpful in differentiating UM from simulating lesions and in the detection of optic nerve involvement or orbital extension.⁴⁴ Biopsies are especially helpful in the diagnosis of suspected intraocular tumors in which it is challenging to obtain a reliable diagnosis after careful fundoscopic examination and adjunctive diagnostic techniques.⁴⁵

Treatment

Eye-conserving therapies and enucleation are the main treatment modalities for primary UM. Enucleation is the traditional treatment option and is nowadays indicated for the treatment of UM in patients with vision loss, for large tumors, tumors in close proximity to the optic disk or which have invaded the optic disk, and cases with extraocular growth.^{46, 47} Small- and medium-sized tumors are mainly treated by plaque brachytherapy using different types of radioactive isotopes, most commonly ¹²⁵Iodium and ¹⁰⁶Ruthenium, which are administered to the tumor by a plaque sutured to the episclera. Brachytherapy and enucleation do not provide a different metastasis rate. The Collaborative Ocular Melanoma Study (COMS) group compared enucleation versus ¹²⁵Iodium brachytherapy for medium- sized tumors and did not find any significant difference in mortality rates at 12- years follow-up:^{48, 49} melanoma-related mortality was 21% in the brachytherapy

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group and 17% in the enucleated patients.⁴⁸ Brachytherapy provides excellent local tumor control, but long-term visual loss is common: the COMS reported substantial impairment of visual acuity within 3 years following ¹²⁵Iodium brachytherapy in 43% to 49% of patients.⁵⁰ Part of the vision loss after brachytherapy is due to the complications of the therapy. Common vision- affecting complications include radiation-induced retinopathy, neovascular glaucoma, and macular edema.⁵¹ Loss of visual acuity occurred mainly in diabetic patients and in patients with thick tumors and retinal detachment. Local tumor recurrence or complications such as neovascular glaucoma may lead to secondary enucleations. A secondary enucleation rate between 12-17% has been reported at 3-5 years follow-up.^{50, 52}

In the past, small UMs were commonly observed for growth and only treated when tumor enlargement was documented.^{53, 54} However, there is a tendency towards earlier treatment since the COMS group reported that 21% of small UMs which were managed by observation showed growth by 2 years and 31% at 5- years follow-up.⁵⁵

Another type of radiotherapy utilized for the treatment of primary UM is proton beam irradiation. Proton beam irradiation could in theory be used for the treatment of all UMs, but is mainly reserved for large tumors in eyes with useful or salvageable vision, as it is a globe-preserving therapy for very large UMs that are not suitable for brachytherapy.⁵⁶ Desjardins et al. treated 2,413 patients by proton beam therapy, and found a 10-year metastasis rate of 27%, compared to 25% and 30% in other studies.⁵⁷

Although recent advances in the treatment of primary UM have resulted in excellent local control and preservation of the eye, survival rates have not

improved significantly.^{12, 58} Damato et al. have shown that timely treatment of the primary tumor may be useful in preventing metastases in small tumors.⁵⁹ Studies on doubling times of UM metastases have indicated that micrometastases probably occur before diagnosis and treatment of the primary UM in a large portion of patients.⁶⁰ Moreover, many patients develop metastases soon after treatment of the primary UM, indicating that subclinical disseminated disease was probably already present at the time of the treatment.⁶¹ In support of this theory, circulating tumor cells have been detected in patients who had no clinical

metastases at diagnosis.⁶² Prognosis and Prognostication

The findings that 1) survival of UM patients has not improved despite advances in

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the local control of the primary tumor; 2) treatment of the primary tumor may only improve survival in small tumors; 3) micrometastases probably occur before diagnosis of the primary tumor, imply that enhancement of the survival of UM patients can mainly be achieved by inventing effective therapeutic modalities for UM metastases. Up to 50% of UM patients eventually develop metastases, usually in the liver, and die because of a lack of effective systemic treatments for

disseminated UM.⁶³ The reported median survival time after detection of metastases is 4 to 15 months.⁶⁴

Various therapeutic options for the treatment of UM metastases are being investigated in clinical trials. Kinase inhibition targeting the MAPK and/or PI3K pathway has been evaluated and proposed as a potential adjuvant therapy to prevent metastatic outgrowth in patients at high risk of developing disseminated disease.⁶⁵ Another focus is immunotherapy which has shown promising results in the treatment of cutaneous melanoma.^{66, 67}

In view of the increasing number of studies evaluating new therapies, determining which patients are at high-risk of developing metastases by reliable

prognostication is relevant for their inclusion in these clinical trials. Identification of high-risk cases is also important for planning of follow-up measures to identify metastases in an early phase and for implementation of adjuvant therapies which may prevent disseminated disease. Furthermore, prognostication allows life- planning in high-risk patients and can be used to reassure those at low-risk of developing metastases.

A variety of clinical, anatomic, histological, and genetic prognostic indicators have been identified in UM and are being utilized in clinical practice. Prognostication by genetic markers has been proven to reliably predict survival in UM and is currently a heavily investigated topic. Genetic prognostication in UM is discussed

extensively in Chapter 2.

THESIS OUTLINE

This thesis is an overview of research performed to better understand the role of genetic and non-genetic factors for prognostication in UM and to identify the function of such factors.

In this introduction, I have provided an overview of the clinical aspects of UM, covering essential topics such as epidemiology, clinical presentation, diagnosis, treatment, and prognostication. Chapter 2 provides a detailed overview of the current status of genetic prognostication in UM, evaluates various types of

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genetic markers, compares genetic tests, and addresses relevant topics related to the application of genetic prognostication in daily clinical practice. In Chapter 3, demographic, anatomic, histological, and genetic prognostic markers that influence survival in long-term surviving patients are addressed, while results demonstrating refinement of prognostication in UM by combining genetic markers and anatomic staging are described in Chapter 4. Since most primary UMs are treated by radiotherapy and chromosome markers are commonly utilized for prognostication, I evaluated the effect of radiation treatment on chromosome testing in UM in Chapter 5.

In Chapter 6, the results of a study evaluating differences in the expression of DNA repair molecules between prognostically-favorable and prognostically- unfavorable UM are presented. Aberrant DNA repair is a hallmark of cancer that plays a role in the development and progression of malignancies and may be used as a target for therapy. I set out to analyze the expression of DNA repair genes in UM, since the role of DNA repair mechanisms in UM has been underexposed.

Similarly, there is lack of knowledge about the role of epigenetic regulators in the pathogenesis of UM. Epigenetic modifications have been shown to contribute to cancer development and progression. I have analyzed the expression levels of a number of epigenetic modifiers in UM. Chapter 7 reports on differences in the expression level of epigenetic markers between UMs with a favorable prognosis and UMs with an adverse prognosis. Chapter 8 provides a summary and general discussion of the findings described in this thesis, and concludes by putting forward future perspectives on genetic prognostication in UM.

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Chapter 2 Genetic Prognostication in Uveal Melanoma

Mehmet Dogrusöz¹, Martine J. Jager¹

1Department of Ophthalmology, Leiden University Medical Center, The Netherlands Published in: Acta Ophthalmol. 2017 Nov 4. [Epub ahead of print]

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24 | C h a p t e r 2 ABSTRACT

Uveal melanoma (UM) is a rare tumor with a high propensity to metastasize.

Although no effective treatment for metastases yet exists, prognostication in UM is relevant for patient counselling, planning of follow-up, and stratification in clinical trials. Besides conventional clinicopathologic characteristics, genetic tumor features with prognostic significance have been identified. Non-random

chromosome aberrations such as monosomy 3 and gain of chromosome 8q are strongly correlated with metastatic risk, while gain of chromosome 6p indicates a low risk. Recently, mutations in genes such as BAP1, SF3B1, and EIF1AX have been shown to be related to patient outcome. Genetics of UM is a rapidly developing field, which not only contributes to the understanding of the pathogenesis of this cancer, but also results in further refinement of prognostication.

Concomitantly, advances have been made in the use of genetic tests. New

methods for genetic typing of UM have been developed. Despite the considerable progress made recently, many questions remain, such as those relating to the reliability of prognostic genetic tests, and the use of biopsied or previously- irradiated tumor tissue for prognostication by genetic testing. In this article, we review genetic prognostic indicators in UM, also comparing available genetic tests, addressing the clinical application of genetic prognostication, and discussing future perspectives for improving genetic prognostication in UM.

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1. INTRODUCTION

Uveal melanoma (UM) arises from melanocytes residing in the uveal tract, which comprises the iris, ciliary body, and choroid. UM accounts for most (85%) ocular melanomas and is the most common primary intraocular malignancy in adults.¹ The annual age-adjusted incidence in the United States is 5.1 per million, with a male-to-female ratio of 1.1:1.² The mean age at diagnosis is 61 years and most patients develop UM after the age of 50.³ Approximately 95% of uveal melanomas occur in Caucasians,³ especially in those with a light iris color, fair skin, propensity to sunburn, and a tendency to develop common/atypical cutaneous nevi and cutaneous freckles.^4-7 Congenital oculodermal melanocytosis (nevus of Ota), which affects 0.04% of the white population,⁸ is associated with a 1 in 400 risk of uveal melanoma. ⁹ Choroidal nevi are estimated to have a 1-in-4300 to 1-in-8845 per year risk of malignant transformation.^10-12 Evidence correlating ultraviolet light exposure with UM is inconclusive. Arc welding has been reported to be a risk factor;¹³ however, welding arcs are also a source of blue light, which has recently been proposed as a risk fator for the development of UM.¹⁴

In the last decades, many advances have been made in the treatment of the primary tumor, which include various forms of radiotherapy, phototherapy and local resection. These eye-sparing methods have largely replaced enucleation, which is now reserved for large UMs, tumors involving the optic nerve and eyes with a poor visual prognosis. Early detection of UM may enhance opportunities for eye-conserving therapy.^{15, 16} Damato et al. have provided tentative evidence that early treatment of tumors may prevent metastatic disease and improve survival in patients with small tumors.¹⁷ However, survival of patients with metastasized UM has not improved because effective treatment is lacking. The overall 10-year metastasis rate is 40% with almost 50% of patients eventually dying from metastases, which usually involve the liver. The median survival time after the diagnosis of metastases ranges from 4 to 15 months.¹⁸

Despite the lack of efficient treatment for metastasized UM, prognostication in UM is valuable since it enables clinicians to reassure patients who have a low risk of metastasis and to target special measures at high-risk patients, who are likely to have clinically undetectable micrometastases at the time of diagnosis of the primary UM.¹⁹ These patients may be stratified in clinical trials to determine the efficacy of adjuvant treatments for UM metastases. Moreover, reliable

prognostication allows risk-based planning of screening for metastases, preventing unnecessary investigations in those with a good prognosis.

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Various patient and tumor characteristics have been identified as survival predictors in UM. For example, the prognosis is better in children than in adults, independently of other risk factors.^{20, 21} Clinical features indicating increased metastatic risk include large tumor size, ciliary body involvement, and extraocular extension.^22-25 These form the basis of the Tumor, Node, Metastasis (TNM) staging system of the American Joint Committee on Cancer (AJCC).²⁶ This prognostication system has recently been validated in large multinational studies.^27,AJCC

Ophthalmic Oncology Task Force ²⁸ Histopathologic predictors of metastasis include epithelioid melanoma cytomorphology,²⁹ high mitotic count,³⁰

lymphocytic infiltration,³¹ extravascular matrix loops ³² and vascular invasion.³³ Early studies of the genetics of UM indicate that non-random alterations of chromosomes 3, 6, and 8 are common and have prognostic significance. Recently, molecular classification of UMs based on gene-expression profiling has been shown to correlate with survival. Several studies have demonstrated that genetic markers have better prognostic accuracy than clinical and histopathologic biomarkers. Although considerable progress has been made in the genetic characterisation of UM, questions remain with regard to the accuracy of markers, the reliability of genetic tests, and the use of biopsy specimens or previously- irradiated tumor tissue for prognostication by genetic testing.

In this article, we overview genetic prognostic indicators in UM, compare current genetic tests, discuss genetic testing in biopsied and irradiated tumors, and propose methods for improving genetic prognostication in UM.

2. GENETIC PROGNOSTIC MARKERS

Genomic instability is one of the hallmarks of cancer.^{34, 35} However, in comparison to cutaneous melanoma and other cancers, UM has relatively few mutations.^36-39 In UM, one can identify recurring non-random chromosome aberrations, which are not the result of chromosomal instability but which are specific alterations that are linked to tumor development and progression.³⁹

2.1 Chromosome alterations

In 1996, a strong association was observed between loss of one copy of

chromosome 3 and the development of metastatic disease.⁴⁰ Several studies had previously reported chromosome 3 aberrations in UM and had described the recurrent and nonrandom nature of these alterations^41-51 Prescher et al. detected monosomy 3 in 56% of a series of 54 UMs and reported a 50% metastasis rate at

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3-years follow-up in patients whose tumor harbored this aberration, while none of the patients with a disomy 3 melanoma had developed metastatic disease.⁴⁰ Subsequent studies in larger cohorts showed monosomy 3 in 25% to 65% of UMs and confirmed the strong association with metastatic disease (Table 1A).^52-62 Monosomy 3 is known to be associated with clinicopathologic features indicative of a poor prognosis, such as large tumor diameter, ciliary body involvement, epithelioid melanoma cytomorphology,^{53, 54, 63} and inflammation.^{64, 65} Nevertheless, monosomy 3 is also predictive of metastatic death independent of

clinicopathologic factors ⁵⁴ and has been shown to be superior to clinicopathologic factors as a prognostic indicator.^{40, 66-68} In UM metastases, the presence of

monosomy 3 has been associated with decreased survival from the time of diagnosis of disseminated disease.⁶⁹

Some studies proposed that complete monosomy is more strongly correlated to metastasic risk than partial monosomy 3.^{56, 70} However, when considering cases with borderline results as ‘normal’ and only defining tumors with definite loss of chromosome 3 as such, Damato and associates reported similar rates of

metastatic death for cases with partial or total loss of chromosome 3,^{55, 71} which was corroborated in a study by Ewens et al.⁵⁹

Another type of loss of heterozygosity (LOH) of chromosome 3, isodisomy 3,⁵¹ which occurs in 5% to 10% of cases, conveys a metastatic risk that is similar to monosomy 3.⁷² Isodisomy is the presence of two identical copies of a

chromosome, both from the same parent. This implies that the pathologic effect of monosomy 3 is not due to haploinsufficiency, but due to complete loss of various tumor suppressor proteins, presumably by mutations on certain loci on the remaining copy of chromosome 3 (see below). It is supposedly this abnormal copy that is duplicated in tumors with isodisomy 3.⁷² The way monosomy 3 affects tumor development and progression has not yet been elucidated. Since

monosomy 3 UMs exhibit a higher level of aneuploidy than disomy 3 tumors, it has been suggested that monosomy 3 leads to increased genomic instability.³⁹ Another chromosome that is frequently altered in UM is chromosome 8.^{42-44, 47} Gain of the long arm of chromosome 8 (8q), which often results from

isochromosome formation, is associated with poor prognosis and occurs in 37% to 63% of primary UM.52, 54, 55, 58, 59, 61, 62, 68 Isochromosome 8q leads to gain of

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Table 1. Frequency of common chromosome aberrations with evident prognostic significance and gene mutations in primary uveal melanoma. Studies are listed in chronologic order. A: chromosome aberrations. B: gene mutations.

A:

B:

Study Chromosome aberration (%)

Loss of 1p Monosomy 3 Gain of 6p Gain of 8q

Prescher et al. 1996 56

Sisley et al. 1997 50 54

Scholes et al. 2003 51

Kilic et al. 2006 24 18 53

Damato et al. 2007 47 37

Damato et al. 2010 34 61 54 63

Shields et al. 2011 25

Thomas et al. 2012 56

Van den Bosch et al. 2012 30 61 42 61

Ewens et al. 2013 19 45 32 51

Ewens et al. 2014 65

Koopmans et al. 2014 31 62 51 58

Dogrusöz et al. 2017 53 47

Range 19-34 25-65 18-54 37-63

Study Gene mutation (%)

GNAQ GNA11 BAP1 SF3B1 EIF1AX Van Raamsdonk et al. 2009 46

Harbour et al. 2010 47

Van Raamsdonk et al. 2010 48 34 Daniels et al. 2012 47 44

Furney et al. 2013 25 58 58 15 8

Harbour et al. 2013 42 52 38 19

Koopmans et al. 2013 50 43

Martin et al. 2013 45 40 21 18

Dono et al. 2014 42 33 32 10 19

Ewens et al. 2014 46 35 50 10 16

Koopmans et al. 2014 47

Decatur et al. 2016 44 44 45 24 17

Moore et al. 2016 43 49 35 18 13

Van de Nes et al. 2016 51

Yavuzyigitoglu et al. 2016 49 45 46 24 21

Range 25-50 33-58 32-58 10-24 8-21

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material because it results in 3 copies of 8q while there is only 1 copy of 8p. An increasing dosage of 8q has been shown to convey an even greater risk of metastatic death.^{52, 73} Gain of 8q commonly accompanies monosomy 3 and the concomitant occurrence of these aberrations is associated with a higher risk of metastasis than either of the aberrations alone.52, 54, 55, 74 We corroborated this in a recently published study in collaboration with the Copenhagen University Hospital Rigshospitalet, in which we reported on combining AJCC staging and chromosome 3 and 8q status to improve prognostication.⁶¹ In the cohort of 470 tumors with known chromosome 3 and 8q status, tumors harboring monosomy 3 as well as chromosome 8q gain showed an increased risk of metastatic death (Figure 1).

Figure 1. Cumulative incidence curves showing death due to uveal melanoma metastases in relation to chromosome 3 and 8q status. Adopted from reference# 61.

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Although less frequently occurring than loss of chromosome 3 and gain of chromosome 8q, loss of the short arm of chromosome 1 (1p) is quite common in UM (19-34%),55, 58, 59, 62, 68 especially in metastasizing tumors (33%).⁷⁵ In keeping with that finding, loss of 1p is associated with monosomy 3 ^{55, 76, 77} and the concurrent loss of 1p and chromosome 3 has been correlated with a decreased disease-free survival.⁷⁸

Chromosome 6 was the first chromosome in which alterations were reported in UM.⁷⁹ The loss of the long arm of chromosome 6 (6q) is more common in metastasizing than in non-metastasizing primary UM,⁷⁵ while, in contrast to all aforementioned chromosomal alterations, gain of the short arm of chromosome 6 (6p) has a protective effect.^{55, 71, 80} However, tumors with a normal chromosome 3 status and normal chromosome 6p status show a better prognosis than those with 6p gain.³⁹ Between 18% and 54% of UM exhibit gain of chromosome 6p,^{55, 58,}

59, 62, 68 which is almost exclusive to monosomy 3, suggesting distinct evolutionary pathways of tumor development.^{76, 81, 82} Although chromosome 8q gain is related to monosomy 3, it is also found in tumors with gain of 6p.³⁹ While it has been hypothesized that monosomy 3 is the first step in the malignant transformation of UM,⁸³ and that 8q gain occurs after monosomy 3 or 6p gain,^{39, 81} a recently

published study reported monosomy 3 heterogeneity in tumors that are

homogeneous for 8q gain; the authors therefore concluded that monosomy 3 is preceded by gain of 8q.⁸⁴ A study by Singh et al. indicated that gain of the telomeric part of 8q has a central role in UM tumorigenesis and reported this aberration in 92% of their studied tumors. Their analysis showed that this aberration is followed by either gain of the centromeric 8q and loss of chromosome 3, or by gain of chromosome 6p, as well as 7q, 11p, and 22q.⁸⁵ 2.2 Gene expression profiling

Since UM is characterized by non-random chromosome aberrations with distinct prognostic implications, it was anticipated that UM could be separated into prognostic groups based on gene expression profiling (GEP). In 2003, Tschentscher et al. performed unsupervised hierarchical cluster analysis of gene expression data on 20 primary tumors using a microarray gene chip of 12,500 probes and defined two distinct molecular classes, correlating with chromosome 3 status.⁸⁶ Zuidervaart et al., in an independent study, performed an mRNA expression array on 12 UM cell lines, and identified four genes that were subsequently used on 19 primary UM samples to separate them into two groups, based on the expression

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of these genes.⁸⁷ A subsequent study of gene expression by Onken et al. in 40 primary UM used a microarray chip containing approximately 45,000 probes and confirmed the clustering of UM into two molecular groups. This study showed that the two observed specific genetic expression profiles (GEP) predicted survival.⁸⁸ Class 1 tumors were found to correlate with a low risk of metastatic death with a 92-month survival rate of 95% as compared to 31% in class 2 UM (the high-risk tumors). In a subgroup analysis of 10 tumors, chromosome 6p gain was found in four of five class 1 tumors and in none of the class 2 tumors, while loss of chromosome 3 occurred in four of five class 2 cases and in none of the class 1 UMs. All class 2 tumors with loss of chromosome 3 also showed gain of chromosome 8q, which was found in only two class 1 tumors.⁸⁸

Recently, class 1 tumors have been subdivided into class 1A (2% 5-year metastatic risk) and class 1B (21% 5-year metastatic risk),⁸⁹ based on the differential

expression of the CDH1 and RAB31 genes.⁹⁰ Class 2 tumors occur more commonly in older patients ⁸⁸ and are related to monosomy 3,⁹¹ greater thickness, epithelioid cell type,⁹² extravascular matrix loops,⁹³ and a higher proliferation rate (Ki-67 score).⁹⁴ Class 2 tumors have been subclustered into class 2A and class 2B tumors.

Class 2B cases harbor a deletion of chromosome 8p that makes the tumors even more aggressive and results in an earlier onset of metastasis compared to class 2A tumors.⁹⁵ Recently, expression of PRAME has been associated with increased metastatic risk in class 1 as well as class 2 tumors.⁹⁰

The association between GEP class and survival has been validated independently in several studies.^{92, 96, 97} For clinical purposes, a practical 15-gene assay based on the 12 most highly discriminating genes and 3 control genes, which can be

performed on small biopsied tumor samples, has been developed,⁹⁸ and validated in a large multicenter study.⁹⁹ It has been claimed that analysis of mRNA is more accurate in prognostication than clinicopathologic parameters or chromosome 3 testing.^{91, 99} However, similar to the original reports of Tschentscher et al.,⁸⁶ Onken et al.,⁸⁸ and van Gils et al.,⁹⁶ the mRNA expression pattern corresponds very strongly with chromosome 3 status.¹⁰⁰ In accordance with earlier reports by Damato’s group on combining clinical, histologic, and genetic predictors to improve prognostication in UM,^{101, 102} a recent study from Harbour’s group ¹⁰³ and another independent study¹⁰⁴ indicated that largest basal diameter provides prognostic information that is independent of GEP (see section 5).

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Unlike cutaneous melanoma, UM does not harbor mutations in BRAF or NRAS genes,^105-109 but instead is characterized by mutations in the GNAQ gene (chromosome 9q) and its paralogue GNA11 (chromosome 19p); these genes encode alpha subunits of the heterotrimeric G proteins associated with the transmembrane G protein-coupled receptors.^110-114 Mutations in these genes are thought to result in the constitutive activation of the mitogen-activated protein kinase (MAPK) pathway and protein kinase C (PKC) pathway, which are involved in cell growth, cell proliferation, differentiation and apoptosis.110, 111, 113, 115, 116 The MAPK pathway is activated in up to 90% of primary UM ¹⁰⁸ and mutations of GNAQ and GNA11 have been reported in 83% to 91% of primary UM, occurring in a mutually-exclusive manner.113, 117, 118 Mutations in GNAQ are reported to occur in 25-50% of tumors, while GNA11-mutant cases account for 33-58% (Table 1B).^60,

111, 113, 117-125

GNAQ and GNA11 mutations are thought to be initiating events in UM pathogenesis since they are present in the majority of UM, regardless of

chromosome aberrations or GEP class, and are also found in benign melanocytic lesions such as blue nevi.110, 111, 113 Van Raamsdonk et al. found a mutation in either GNAQ or GNA11 in 61% of the 139 blue nevi they have tested,¹¹³ and reported an 83% mutation frequency for GNAQ in 29 tested blue nevi in an earlier study.¹¹¹ Although most studies could not find a correlation between GNAQ or GNA11 and survival,113, 118, 126 a recent study by Griewank et al. reported a predominance of GNA11 mutations in UM metastases, and a poorer disease- specific survival of GNA11-mutant tumors in a cohort of 30 UM patients with metastases.¹²⁷ In 101 UMs treated by primary enucleation in the LUMC, we found monosomy 3 in 70% of GNA11-mutant UMs (n=53) versus 48% in GNAQ-mutant UMs (n=48) (Pearson’s chi-squared test, p=0.03) (unpublished data). Although we noticed a trend towards worse survival for GNA11-mutant tumors compared to GNAQ-mutant cases, this difference was not signicant (log-rank test, p=0.27) (unpublished data).

As mentioned above, the strong correlation between loss of heterozygosity of chromosome 3 and an adverse prognosis raised the suspicion that loss of function of tumor suppressor genes on chromosome 3 may result in a malignant

phenotype. Early efforts to identify the critical region of chromosome 3 yielded varying results.^128-130 Blasi et al. found a translocation involving chromosome region 3p13 as the only clinical aberration in a primary UM cell culture and

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suggested that this region could harbor a pathogenically-relevant tumor suppressor gene.¹²⁸ Tschentscher et al. investigated partial deletions of

chromosome 3 and found two regions (3q24-26 and 3p25) that were frequently lost.¹²⁹ A study by Parrella et al. identified the same region (3p25.1-25.2), and overlapping results were reported by Cross et al. and van Gils et al., who also speculated on a segment (3p12-3p14) similar to the one addressed earlier by Blasi et al. 96, 128, 130, 131

In 2010, inactivating hemizygous somatic mutations of the BAP1 (BRCA1-

associated protein 1) gene on chromosome 3p21.1 were identified in 47% (27/57) of cases. BAP1 mutation was found in most metastasizing UMs, occurring in 84%

(26/31) of class 2 tumors and in only 4% (1/26) of class 1 cases.¹³². Subsequent studies showed that inactivating mutations of BAP1 occur in 32-58% of primary UM.60, 62, 119-122, 124, 125, 133 Mutation of BAP1 is also strongly correlated with

chromosome 3 status, ocurring in 89% of monosomy 3 tumors and in no disomy 3 tumors, in a cohort of 66 Ums.¹³³

Loss of BAP1 gene expression has been shown to correlate well with the lack of BAP1 protein expression, which has been proposed as a clinically valuable

prognostic tool.62, 100, 133-135 Metastases arise when there is a combination of loss of one chromosome 3 and a mutation in the BAP1 gene on the other chromosome, leading to loss of expression of BAP1.^{62, 100}

The BAP1 protein is a ubiquitin carboxyterminal enzyme that affects the activity of other proteins through deubiquitination. For example, it regulates gene

expression epigenetically by removing ubiquitin molecules from histone H2A. It has been demonstrated that loss of BAP1 function leads to the loss of the melanocytic cell phenotype and loss of differentiation in UM.¹³⁶

Germline mutations in BAP1 ^{137, 138} have been identified in 2-3% of UM

patients.^139-141 These patients tend to have a family history of UM. A recent study reported BAP1 germline mutations in approximately 20% of familial cases of UM.¹⁴² Patients with BAP1 germline mutations have larger tumors, with more common ciliary body involvement, both of which are related to a higher risk of metastasis.¹⁴⁰ These mutations may be present in UM occurring at a younger age.¹⁴³ In addition, patients with germline BAP1 mutations are at higher risk of other cancers such as lung adenocarcinoma, renal cell carcinoma, meningioma, and malignant mesothelioma,137, 144-146 prompting the need for treating physicians to recognize familial cases of UM and to identifying patients with germline BAP1 mutations.

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In contrast to BAP1, mutations in the SF3B1 (splicing factor 3 subunit B1) gene on chromosome 2q are associated with favorable prognostic parameters such as younger age at diagnosis and fewer epithelioid cells, whilst being inversely associated with adverse prognostic features such as monosomy 3 and the class 2 gene expression profile.¹²³ Patients with mutations in this gene account for 10% to 24% of UM cases.60, 119-125 In the study by Furney et al., patients with SF3B1

mutations showed a better prognosis than patients with SF3B1-wildtype tumors,¹²¹ while in another study significance could not be reached ¹²³ and in a study with a relatively short follow-up (48 months) no association with metastatic disease was reported.⁶⁰ In a long-term study by Yavuzyigitoglu et al. using whole- exome sequencing, an association of mutated SF3B1 and favorable prognosis was observed in the overall group (n=133, 32 SF3B1-mutant) during the first few years of follow-up; however, this difference was less evident at longer follow-up since patients with SF3B1 were noticed to develop metastases at a later stage. Within the disomy 3 cohort, patients with SF3B1 mutations had an increased metastasic risk when compared to patients without this mutation and developed metastases at a median follow-up of 8.2 years. SF3B1 mutation was therefore correlated with late-onset metastasis and was the only parameter independently associated with worse survival in disomy 3 tumors in the multivariate analysis. Most (11/14) disomy 3 patients who developed metastases had an SF3B1 mutation, while BAP1 mutations were found in two other disomy 3 patients who developed

metastases.¹²⁵ These mutations were missense mutations and the tumors stained positively for BAP1 using immunohistochemistry. Although it may be assumed that a nonfunctional protein is produced, this should be validated by functional assays.

Mutations in the EIF1AX (eukaryotic translation initiation factor 1A, X-linked) gene on chromosome Xp are found in 8% to 21% of primary UMs and are associated with a decreased risk of metastasis.60, 119-121, 123-125 Ewens et al. reported a 10-fold lower metastasic risk for disomy 3/BAP1-wild type/EIF1AX-mutant tumors, when compared to disomy 3/BAP1-wild type/EIF1AX-wild type cases.⁶⁰ The association of EIF1AX mutations and a favorable clinical outcome was confirmed in two recently published studies.^{119, 125} Together, these reports show that mutations in BAP1, SF3B1 and EIF1AX occur in a mutually exclusive manner, which has been underlined by a study that reported on the results of whole-genome sequencing in 33 samples.¹⁴⁷ Moreover, mutations in these three genes are associated with differing risks of developing metastasis. Tumors with BAP1 mutations show a high

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and early metastatic risk whereas tumors with mutated SF3B1 are associated with late-onset metastasis and EIF1AX-mutant tumors have a very low metastatic risk.¹²⁵

3. GENETIC TESTS

Diverse genetic techniques, such as karyotyping, fluorescence in situ hybridization (FISH), multiplex ligation-dependent probe amplification (MLPA), array-based comparative genomic hybridization (aCGH), single-nucleotide polymorphism (SNP) assay, and GEP are commonly utilized to determine genomic tumor characteristics with prognostic value in UM. Relevant aspects to take into consideration with regard to the application of a certain test are the type of tumor specimen (fresh tumor tissue/frozen/formalin-fixed paraffin embedded), the available genetic material from the tumor specimen (DNA/RNA), the prognostic accuracy, and the costs of the test. In this section, we discuss briefly the most important tests that can be utilized for genetic prognostication in UM and mention their respective advantages and disadvantages (Table 2).

Initial studies reporting on the prognostic value of aberrations in chromosomes 3 and 8 ^40-43 used karyotyping of short-term cultured UM cells, which was also utilized in later studies to further characterize UM cytogenetically.^{68, 148} The advantage of karyotyping is that it provides information on all chromosomes in a single assay and allows the identification of structural and balanced chromosome abnormalities, in addition to numerical changes. However, tumor specimens must be fresh since viable dividing cells are required. Furthermore, this method is labor-intensive test and has to be performed by an experienced cytogeneticist.

Another disadvantage of karyotyping is that it can only reliably detect gross aberrations due to its overall low resolution of approximately 5 to 10 Mega base pairs (Mbp).^149-151 Kilic et al. have reported a 100% 10-year mortality in patients with loss of chromosome 3p detected by karyotyping, and a 30% mortality rate in patients without this aberration.⁶⁸ The relatively high percentage of mortality in patients without detected loss of chromosome 3 may be explained by the low sensitivity of karyotyping in detecting LOH in cases of isodisomy 3 (copy-neutral LOH).⁵¹

Another approach to chromosomal testing is FISH, which can be performed on aged, frozen and paraffin-embedded specimens as well as fresh samples. FISH uses a technique where a specific colored probe is used that binds

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Table 2. Main advantages and limitations of genetics tests commonly utilized in UM prognostication.

Genetic test Main advantages Main limitations

Karyotyping - information on all chromosomes in single assay

- identification structural and balanced chromosome abnormalities

- fresh tumor specimens with viable dividing cells needed - labor-intensive

- experienced cytogeneticist required - low resolution

(only reliable for gross aberrations) - prone to sampling error

(tumor heterogeneity) - can not detect isodisomy 3 FISH - can be performed on fixed samples

- relatively easy technique - only identification targeted (regions of) chromosomes - can not detect isodisomy 3 - prone to sampling error (tumor heterogeneity) aCGH - can be performed on fixed samples

- provides copy numbers of all chromosomes

- high resolution (can detect smaller aberrations than karyotyping and FISH)

- can not detect isodisomy 3 - prone to sampling error (tumor heterogeneity)

SNP - can be performed on fixed samples - high resolution

- detects isodisomy 3 - relatively inexpensive

- prone to sampling error (tumor heterogeneity)

MSA - can be performed on fixed samples - detects isodisomy 3

- inexpensive

- prone to sampling error (tumor heterogeneity) - low resolution MLPA - can be performed on fixed samples

- suitable for small samples (biopsies) - detects aberrations of chromosomes 1p,3,6, and 8 in single reaction - relatively inexpensive

- prone to sampling error (tumor heterogeneity) - can not detect isodisomy 3

GEP - can be performed on fixed samples - gene expression information on all chromosomes