Cancer immunotherapies utilizing immune checkpoint blockade (ICB) therapy targeting CTLA-4 and PD-1/PD-L1 relieve tumor-induced immune suppression and induce durable tumor regression. The use of... Show moreCancer immunotherapies utilizing immune checkpoint blockade (ICB) therapy targeting CTLA-4 and PD-1/PD-L1 relieve tumor-induced immune suppression and induce durable tumor regression. The use of ICB therapy have demonstrated remarkable therapeutic efficacy in a proportion of patients with melanoma. However, still a substantial percentage of patients does not respond (durable) to ICB treatment and many questions remain. Therefore, in this thesis, the aim is to improve our understanding of ICB efficacy. We demonstrate the promise of neoadjuvant ICB therapy (approach in which ICB therapy is applied before surgery) and analyze different cohorts of melanoma patients. This results in the identification of several markers that are associated with prognosis, including IFN-y related gene signature score, Batf3 dendritic cell associated gene signature score, tumor mutational burden and systemic LRG1 expression. These markers can potentially be targeted and might facilitate rational combination therapies that can boost the efficacy of ICB therapy. For this purpose, we perform a repurposing compound screen that targets antigen cross-presentation. Togethers, this work increases our understanding of factors that determine ICB therapy efficacy and toxicity, with the goal to identify novel strategies to improve outcome of melanoma patients in a rationale and personal manner. Show less
Uveal melanoma (UM) is a rare ocular tumor. Up to 50% of the patients develop distant metastases predominantly targeting the liver. The median survival after diagnosis of patients with hepatic... Show moreUveal melanoma (UM) is a rare ocular tumor. Up to 50% of the patients develop distant metastases predominantly targeting the liver. The median survival after diagnosis of patients with hepatic metastases is approximately 4-6 months and hardly increased in the past decades due to lack of novel effective therapeutic options. Within the scope of this thesis we investigated the signaling landscape of metastatic UM and searched for novel avenues of therapy. In Chapter 2 we demonstrate that combinations of the multitarget drug Trabectedin with either the CK2/Clk double-inhibitor Silmitasertib or with the c-MET/TAM receptor inhibitors show synergistic growth inhibitory effects and induce apoptosis of UM cells in vitro. Chapter 3 describes the application of a CRISPR-Cas9 synthetic lethality screen for identification of molecular targets whose inhibition synergistically enhances the effect of the mTOR inhibitor everolimus in UM cells. In Chapter 4 we show that the combination of genetic depletion YAP1/TAZ together with Mcl-1 inhibition resulted in a synergistic inhibitory effect on the viability of UM cell lines. In Chapter 5 we analyzed the phospho-proteome of two UM metastatic cell lines and a primary tumor cell line from the same individual, and studied the role of MARK3 in YAP1/TAZ signaling. Show less
The aim of this thesis was to develop novel treatment strategies for different types of eye melanoma utilizing zebrafish models. We first establish orthotopic and ectopic xenograft models for uveal... Show moreThe aim of this thesis was to develop novel treatment strategies for different types of eye melanoma utilizing zebrafish models. We first establish orthotopic and ectopic xenograft models for uveal and conjunctival melanoma by engraftment of the immortalized cells derived from these tumors into zebrafish embryos. Next, we expanded these models with spheroids and zebrafish patient-derived xenografts for pre-clinical, personalized screening of anti-uveal melanoma drug responses. We demonstrated that these models can be harnessed to explore the in vivo interactions of the tumor cells with blood vessels and macrophages leading to angiogenic response. We finally apply the conjunctival melanoma model to clarify the inhibitory effects of ginsenosides and correlate their structures with potential antitumoral mechanisms. Show less
PurposeMRI is increasingly used in the diagnosis and therapy planning of uveal melanoma (UM). In this prospective cohort study, we assessed the radiological characteristics, in terms of anatomical... Show morePurposeMRI is increasingly used in the diagnosis and therapy planning of uveal melanoma (UM). In this prospective cohort study, we assessed the radiological characteristics, in terms of anatomical and functional imaging, of UM after ruthenium-106 plaque brachytherapy or proton beam therapy (PBT) and compared them to conventional ultrasound.MethodsTwenty-six UM patients were evaluated before and 3, 6 and 12 months after brachytherapy (n = 13) or PBT (n = 13). Tumour prominences were compared between ultrasound and MRI. On diffusion-weighted imaging, the apparent diffusion value (ADC), and on perfusion-weighted imaging (PWI), the time-intensity curves (TIC), relative peak intensity and outflow percentages were determined. Values were compared between treatments and with baseline.ResultsPre-treatment prominences were comparable between MRI and ultrasound (mean absolute difference 0.51 mm, p = 0.46), but larger differences were observed post-treatment (e.g. 3 months: 0.9 mm (p = 0.02)). Pre-treatment PWI metrics were comparable between treatment groups. After treatment, brachytherapy patients showed favourable changes on PWI (e.g. 67% outflow reduction at 3 months, p < 0.01). After PBT, significant perfusion changes were observed at a later timepoint (e.g. 38% outflow reduction at 6 months, p = 0.01). No consistent ADC changes were observed after either treatment, e.g. a 0.11 x 10(-3)mm(2)/s increase 12 months after treatment (p = 0.15).ConclusionMR-based follow-up is valuable for PBT-treated patients as favourable perfusion changes, including a reduction in outflow, can be detected before a reduction in size is apparent on ultrasound. For brachytherapy, a follow-up MRI is of less value as already 3 months post-treatment a significant size reduction can be measured on ultrasound. Show less
Ocular melanoma and colorectal carcinoma are two malignancies with a predilection for metastasizing to the liver. Patients with liver-only or liver-dominant metastatic disease might be eligible for... Show moreOcular melanoma and colorectal carcinoma are two malignancies with a predilection for metastasizing to the liver. Patients with liver-only or liver-dominant metastatic disease might be eligible for locoregional or so-called liver-directed therapy. Liver-directed therapies include surgery and thermal ablation, as well as various arterial therapies such as percutaneous hepatic perfusion with melphalan (M-PHP). Although M-PHP is well-tolerated by most patients, hematologic events due to bone marrow suppression were quite common in M-PHP using the first-generation filter. In an attempt to reduce bone marrow suppression by increasing the filter extraction rate, a new second-generation filter (GEN 2 filter) was developed and became commercially available in 2012. In this thesis, it was demonstrated that M-PHP using the GEN 2 filter has an acceptable safety and toxicity profile and it seems to reduce hematologic toxicity when compared to M-PHP with a first-generation filter. This thesis contributes to the scientific evidence showing that M-PHP using the GEN 2 filter is an effective treatment for liver metastases from ocular melanoma. In contrast, M-PHP seems to have no additional value in the current treatment of unresectable liver metastases from colorectal carcinoma. Show less
Jaarsma-Coes, M.G.; Ferreira, T.A.; Marinkovic, M.; Vu, T.H.K.; Vught, L. van; Haren, G.R.V.; ... ; Beenakker, J.W.M. 2023
Objective: Conventionally, ocular proton therapy (PT) is planned using measurements obtained by an ophthalmologist using ultrasound, fundoscopy, biometry, and intraoperative assessments. Owing to... Show moreObjective: Conventionally, ocular proton therapy (PT) is planned using measurements obtained by an ophthalmologist using ultrasound, fundoscopy, biometry, and intraoperative assessments. Owing to the recent advances in magnetic resonance imaging (MRI) of uveal melanoma (UM), it is possible to acquire high-resolution 3-dimensional images of the eye, providing the opportunity to incorporate MRI in ocular PT planning. In this study, we described how these measurements can be obtained using MRI, compared the MRI-based measurements with conventional ophthalmic measurements, and identified potential pitfalls for both modalities. Design: Cross-sectional study. Subjects: Data from 23 consecutive patients with UM treated with PT were retrospectively evaluated. Methods: Magnetic resonance imaging-based measurements of axial length, tumor height and basal diameter, and marker-tumor distances were compared with the conventional ophthalmic measurements, and discrepancies were evaluated in a multidisciplinary setting. Main Outcome Measures: Tumor prominence and basal diameters on MRI and ultrasound, axial length on MRI and biometry, tumor-marker distances on MRI and measured intraoperatively. Results: The mean absolute differences of the tumor height and basal diameter measurements between ultrasound and MRI were 0.57 mm and 1.44 mm, respectively. Larger absolute differences in height and basal diameter were observed when the full tumor extent was not visible on ultrasound (0.92 mm and 1.67 mm, respectively) compared with when the full tumor extent was visible (0.44 mm and 1.15 mm, respectively). When the full tumor was not visible on ultrasound, MRI was considered more reliable. Tumor-marker distances measured using MRI and intraoperative techniques differed < 1 mm in 55% of the markers. For anteriorly located and mushroom-shaped tumors (25% of the markers), MRI provided more accurate measurements. In flat UM (15% of the markers), however, it was difficult to delineate the tumor on MRI. The mean absolute difference in axial length between optical biometry and MRI was 0.50 mm. The presence of the tumor was found to influence optical biometry in 15 of 22 patients; the remaining patients showed a better agreement (0.30 mm). Magnetic resonance imaging-based biometry was considered more reliable in patients with UM. Conclusions: Magnetic resonance imaging allowed for the 3-dimensional assessment of the tumor and surrounding tissue. In specific patients, it provided a more reliable measurement of axial length, tumor dimensions, and marker-tumor distances and could contribute to a more accurate treatment planning. Never-theless, a combined evaluation remains advised, especially for flat UM. Show less
In this thesis I investigate new ways to use MRI (magnetic resonance imaging) for the diagnosis, treatment and follow-up of uveal melanoma (UM) patients, mainly in relation to the planning of... Show moreIn this thesis I investigate new ways to use MRI (magnetic resonance imaging) for the diagnosis, treatment and follow-up of uveal melanoma (UM) patients, mainly in relation to the planning of proton beam therapy. Proton beam therapy is performed while sitting whereas MRI scans are scanned in prone position. In chapter 2 I have shown that the shape of the eye and tumor are not affected by the change in position. During treatment planning the tumor shape needs to be determined. This can be done by drawing the tumor on MRI. In chapter 3 I have shown that the variance between segmentations performed by different doctors are on average smaller then 0.4mm. As MRI based planning is not yet available for UM patients we have developed an MRI protocol to support the current model based treatment planning software with MRI based measurements. In chapter 4 I compare these MRI based measurements with conventional measurements and show that MRI measurements are comparable and sometimes even better. A common side effect of UM is retinal detachment. This is sometimes treated with silicon oil. Unfortunately ultrasound imaging is not possible in these patients. In chapter 5 I describe and evaluate a MRI protocol to imaging these tumors with MRI. Finally, MRI can also provide information about tissue and functional characteristics. In chapter 6 I present a method to overcome eye specific challenges in the quantitative analysis of perfusion weighted MRI. Show less
Background and Purpose: Three-dimensional (3D) Magnetic Resonance Imaging (MRI) is increasingly used to complement conventional two-dimensional ultrasound in the assessment of tumour dimension... Show moreBackground and Purpose: Three-dimensional (3D) Magnetic Resonance Imaging (MRI) is increasingly used to complement conventional two-dimensional ultrasound in the assessment of tumour dimension measurement of uveal melanoma. However, the lack of definitions of the 3D measurements of these tumour dimensions hinders further adaptation of MRI in ocular radiotherapy planning. In this study, we composed 3D MR-based definitions of tumour prominence and basal diameter and compared them to conventional ultrasound. Materials and methods: Tumours were delineated on 3DT2 and contrast-enhanced 3DT1 (T1gd) MRI for 25 pa-tients. 3D definitions of tumour prominence and diameter were composed and evaluated automatically on the T1gd and T2 contours. Automatic T1gd measurements were compared to manual MRI measurements, to auto-matic T2 measurements and to manual ultrasound measurements. Results: Prominence measurements were similar for all modalities (median absolute difference 0.3 mm). Auto-matic T1gd diameter measurements were generally larger than manual MRI, automatic T2 and manual ultra-sound measurements (median absolute differences of 0.5, 1.6 and 1.1 mm respectively), mainly due to difficulty defining the axis of the largest diameter. Largest differences between ultrasound and MRI for both prominence and diameter were found in anteriorly located tumours (up to 1.6 and 4.5 mm respectively), for which the tumour extent could not entirely be visualized with ultrasound. Conclusions: The proposed 3D definitions for tumour prominence and diameter agreed well with ultrasound measurements for tumours for which the extent was visible on ultrasound. 3D MRI measurements generally provided larger diameter measurements than ultrasound. In anteriorly located tumours, the MRI measurements were considered more accurate than conventional ultrasound. Show less
Purpose The aim of this study was to identify positive predictors for survival in uveal melanoma (UM) patients treated with percutaneous hepatic perfusion with melphalan (M-PHP), by retrospectively... Show morePurpose The aim of this study was to identify positive predictors for survival in uveal melanoma (UM) patients treated with percutaneous hepatic perfusion with melphalan (M-PHP), by retrospectively pooling data from three centers.Materials and Methods Retrospective analysis including patients ( >= 18 years) treated with M-PHP between February 2014 and December 2019 for unresectable liverdominant or liver-only metastases from UM. Predictors for OS were assessed using uni- and multivariate analyses. Other study outcome measures were response rate, progression-free survival (PFS), liver progression-free survival (LPFS), overall survival (OS) and complications according to CTCAEv5.0.Results In total, 101 patients (47.5% males; median age 59.0 years) completed a minimum of one M-PHP. At a median follow-up time of 15.0 months, complete response (CR), partial response (PR), stable disease (SD) and progressive disease were seen in five (5.0%), 55 (54.5%), 30 (29.7%) and 11 (10.9%) patients, respectively, leading to a 89.1% disease control rate. Median PFS, LPFS and OS were 9.0, 11.0 and 20.0 months, respectively. Survival analyses stratified for radiological response demonstrated significant improved survival in patients with CR or PR and SD category. Treatment of the primary tumor with radiotherapy, >= 2 M-PHP and lactate dehydrogenase (LDH) < 248 U/L were correlated with improved OS. Thirty-day mortality was 1.1% (n = 2). Most common complication was hematological toxicity (self-limiting in most cases).Conclusion M-PHP is safe and effective in patients with UM liver metastases. Achieving CR, PR or SD is associated with improved survival. Primary tumor treatment with radiotherapy, normal baseline LDH and > 1 M-PHP cycles are associated with improved OS. Show less
Piaggio, F.; Croce, M.; Reggiani, F.; Monti, P.; Bernardi, C.; Ambrosio, M.; ... ; Amaro, A. 2022
Background and aim of the study: Mutations in the G alpha-genes GNAQ and GNA11 are found in 85-90% of uveal melanomas (UM). Aim of the study is to understand whether the mutations in both genes... Show moreBackground and aim of the study: Mutations in the G alpha-genes GNAQ and GNA11 are found in 85-90% of uveal melanomas (UM). Aim of the study is to understand whether the mutations in both genes differentially affect tumor characteristics and outcome and if so, to identify potential mechanisms. Methods: We analyzed the association between GNAQ and GNA11 mutations with disease specific survival, gene expression profiles, and cytogenetic alterations in 219 UMs. We used tandem-affinity-purification, mass spectrometry and immunoprecipitation to identify protein interaction partners of the two G-proteins and analyzed their impact on DNA-methylation. Results: GNA11 mutation was associated with: i) an increased frequency of loss of BRCA1- associated protein 1 (BAP1) expression (p = 0.0005), ii) monosomy of chromosome 3 (p < 0.001), iii) amplification of chr8q (p = 0.038), iv) the combination of the latter two (p = 0.0002), and inversely with v) chr6p gain (p = 0.003). Our analysis also showed a shorter disease-specific survival of GNA11-mutated cases as compared to those carrying a GNAQ mutation (HR = 1.97 [95%CI 1.12-3.46], p = 0.02). GNAQ and GNA11 encoded G-proteins have different protein interaction partners. Specifically, the Tet Methylcytosine Dioxygenase 2 (TET2), a protein that is involved in DNA demethylation, physically interacts with the GNAQ protein but not with GNA11, as confirmed by immunoprecipitation analyses. High risk UM cases show a clearly different DNA-methylation pattern, suggesting that a different regulation of DNA methylation by the two G-proteins might convey a different risk of progression. Conclusions: GNA11 mutated uveal melanoma has worse prognosis and is associated with high risk cytogenetic, mutational and molecular tumor characteristics that might be determined at least in part by differential DNA-methylation. (C) 2022 Elsevier Ltd. All rights reserved. Show less
Ocular melanoma is a rare disease that originates from melanocytes in the eye. It is the most prevalent primary ocular malignancy in adults, and has a high metastatic rate. Two important questions... Show moreOcular melanoma is a rare disease that originates from melanocytes in the eye. It is the most prevalent primary ocular malignancy in adults, and has a high metastatic rate. Two important questions for good patient care are: 1) How to differentiate between (benign) nevi, and (malignant) melanoma?, and 2) How to treat this tumor best, particularly in cases with metastases?This thesis addresses two types of ocular melanoma: melanoma of the internal parts of the eye (uveal melanoma) and melanoma of the mucous membrane covering the eye (conjunctival melanoma). This thesis combines patient-related projects with projects from the lab.With new imaging techniques we demonstrate that oxygen values differ in eyes with melanoma compared to other eyes including those with a nevus. We use OCT-angiography to depict tumour vessels non-invasively in conjunctival and iris lesions. These two techniques may be used in the future to differentiate lesions, and to monitor patients after treatment.With studies in the lab we show that new drugs (immunotherapy) that are recently used in cutaneous melanoma, can also be used to treat conjunctival melanoma. We show that vascular growth in uveal melanoma is related to other (genetic and immunologic) characteristics, providing new clues for therapy. Show less
Purpose: Uveal melanoma (UM) is a rare disease and the most common primary intraocular malignancy in adults, with a high risk of metastases. Reliable prognostication systems are based on anatomic... Show morePurpose: Uveal melanoma (UM) is a rare disease and the most common primary intraocular malignancy in adults, with a high risk of metastases. Reliable prognostication systems are based on anatomic features, as in the tumor-node-metastasis staging of the American Joint Committee on Cancer (AJCC) system, or on genetic information, as in The Cancer Genome Atlas (TCGA) system. Prior evidence suggests that combining both systems may be beneficial. We evaluated the benefit of combining the TCGA and AJCC systems in a large cohort of patients.Design: Retrospective case series of patients with UM.Participants: Nine hundred seventy-nine patients with a choroidal or ciliary body melanoma treated at the Wills Eye Hospital between 1998 and 2020, 94% of whom received eye-sparing treatment.Methods: Tumors were classified into 4 TCGA groups based on chromosome copy number: A (disomy 3, normal 8q), B (disomy 3, any 8q gain), C (monosomy 3, 1 extra copy of 8q), and D (monosomy 3, multiple 8q gain). The eighth edition of the AJCC staging manual was used for AJCC staging. Cox regression and the log-rank test were used for survival analysis.Main Outcome Measure: Metastasis-free survival.Results: Combining information of the 2 systems improved prognostication in intermediate groups: in TCGA group C, we saw an increased rate of metastasis in AJCC stage III (28%) compared with stage 11 (8.9%); the same was seen in AJCC stage II, going from TCGA group C (8.9%) to group D (46%), and in AJCC stage III, going from group C (28%) to group D (49%). In patients with AJCC stage II or III disease, loss of chromosome 3 and gain of 8q (TCGA groups C and D) significantly worsened the prognosis, with multiple 8q gain (TCGA group D) having a greater impact.Conclusions: Combining information from AJCC stages and TCGA groups yields a better predictive power even in this set of relatively small tumors. We propose that physicians take both systems into account whenever possible, especially in moderate-risk groups. (C) 2021 by the American Academy of Ophthalmology. Show less
Part I - Hepatic perfusion for the treatment of unresectable liver metastasesBecause the majority of metastasized uveal melanoma (UM) patients have unresectable liver only metastases, locoregional... Show morePart I - Hepatic perfusion for the treatment of unresectable liver metastasesBecause the majority of metastasized uveal melanoma (UM) patients have unresectable liver only metastases, locoregional therapy was developed. In this thesis percutaneous hepatic perfusion (PHP) is described as a treatment for these patients. During this procedure, the chemotherapeutic agent is infused in the hepatic artery and thereby delivered to the liver and metastases directly. Via a veno-venous filtration system, the chemotherapeutic agent is filtered before it reaches the systemic circulation. In this thesis a clinical study was described treating 20 UM patients with metastases confined to the liver with PHP. It was concluded that the results PHP outbalanced the (minimal) toxicity for patients with uveal melanoma metastases.Part II - Tailored care for patients with pancreatic cancerThe poor prognosis of pancreatic cancer did not change much over the last decades, despite the improvements in treatment modalities. Previous studies have reported variations in incidence and mortality in pancreatic cancer between countries worldwide and European countries. A collaboration was initiated across Europe to compare patterns of care and identify best practices for pancreatic cancer care. A core dataset was identified to identificate differences in age, gender, incidence, tumour stage and differences in treatment strategies. To identify any differences in treatment and/or survival of elderly patients with pancreatic cancer, a comparison was performed of data on geriatric pancreatic cancer care and survival with data from the Netherlands and a special ‘Senior Adult Oncology Program’. Show less
Purpose To evaluate the magnetic resonance imaging (MRI) characteristics of uveal melanoma (UM), to compare them with fundoscopy and ultrasound (US), and to validate them with histopathology.... Show morePurpose To evaluate the magnetic resonance imaging (MRI) characteristics of uveal melanoma (UM), to compare them with fundoscopy and ultrasound (US), and to validate them with histopathology. Methods MR images from 42 UM were compared with US and fundoscopy, and on 14 enucleated cases with histopathology. Results A significant relationship between the signal intensity on T1 and pigmentation on histopathology was found (p=0.024). T1 hyperintense UM were always moderately or strongly pigmented on histopathology, while T1-hypointense UM were either pigmented or non-pigmented. Mean apparent diffusion coefficient (ADC) of the UM was 1.16 +/- 0.26 x 10(-3) mm(2)/s. Two-thirds of the UM had a wash-out and the remaining a plateau perfusion time-intensity curve (TIC). MRI was limited in evaluating the basal diameter of flat tumors. US tends to show larger tumor prominence (0.5mm larger, p=0.008) and largest basal diameter (1.4mm larger, p<0.001). MRI was good in diagnosing ciliary body involvement, extrascleral extension, and optic nerve invasion, but limited on identifying scleral invasion. An increase of tumor prominence was associated with lower ADC values (p=0.030) and favored a wash-out TIC (p=0.028). An increase of tumor ADC correlated with a plateau TIC (p=0.011). Conclusions The anatomical and functional MRI characteristics of UM were comprehensively assessed. Knowing the MRI characteristics of UM is important in order to confirm the diagnosis and to differentiate UM from other intra-ocular lesions and because it has implications for treatment planning. MRI is a good technique to evaluate UM, being only limited in case of flat tumors or on identifying scleral invasion. Show less
Objective Dynamic contrast enhanced (DCE)-MRI is currently not generally used for intraocular masses as lesions are small, have an inhomogeneous T-1 and the eye is prone to motion. The aim of this... Show moreObjective Dynamic contrast enhanced (DCE)-MRI is currently not generally used for intraocular masses as lesions are small, have an inhomogeneous T-1 and the eye is prone to motion. The aim of this paper is to address these eye-specific challenges, enabling accurate ocular DCE-MRI. Materials & methods DCE-MRI of 19 uveal melanoma (UM) patients was acquired using a fat-suppressed 3D spoiled gradient echo sequence with TWIST (time-resolved angiography with stochastic trajectories sequence). The analysis consisted of a two-step registration method to correct for both head and eye motion. A T-1 map was calculated to convert signal intensities to concentrations. Subsequently, the Tofts model was fitted voxel wise to obtain K-trans and v(e). Results Registration significantly improved the concentration curve quality (p < 0.001). The T-1 of melanotic lesions was significantly lower than amelanotic lesions (888 ms vs 1350 ms, p = 0.03). The average achieved B-1(+) in the lesions was 91%. The average K-trans was 0.46 min(-1) (range 0.13-1.0) and the average v(e) was 0.22 (range 0.10-0.51). Conclusion Using this eye-specific analysis, DCE of intraocular masses is possible which might aid in the diagnosis, prognosis and follow-up of UM. Show less
As HLA Class I expression is an important target for cytotoxic T cells but an in inhibitor of NKcells, we were interested in the regulation of its expression.We review HLA expression in UM, how it... Show moreAs HLA Class I expression is an important target for cytotoxic T cells but an in inhibitor of NKcells, we were interested in the regulation of its expression.We review HLA expression in UM, how it is involved in the inflammatory phenotype, how it is regulated and how putative treatments might be effective in its expression.We investigate the potential role of the NFkB pathway in the regulation of inflammation in UM and its potential association with HLA Class I expression.In order to increase our understanding for the reason behind the elevated HLA Class I expression in UM tumours, we investigate the involvement of epigenetics. We focus on a set of epigenetic enzymes called histone deacetylases and report that these regulators are highly expressed in Monosomy 3 UM.We wonder whether HDAC expression is influenced by the presence of infiltrating lymphocytes and macrophages.We focus on miRNA’s as another set of epigenetic regulators of inflammation. We investigate the potential relation of a set of 125 miRNA’s with HLA Class I expression and the presence of an infiltrate in UM and report two patterns of miRNA expression.We study the LAG3 immune checkpoint in UM tumours. As immune checkpoints might be responsible for the T cell exhaustion which is observed in UM, we investigate the involvement of LAG in prognostication and study how LAG3 and its ligands are distributed among different UM tumours. Show less
Lange, M.J. de; Nell, R.J.; Velden, P.A. van der 2021
Here, we discuss the presence and roles of heterogeneity in the development of uveal melanoma. Both genetic and cellular heterogeneity are considered, as their presence became undeniable due to... Show moreHere, we discuss the presence and roles of heterogeneity in the development of uveal melanoma. Both genetic and cellular heterogeneity are considered, as their presence became undeniable due to single cell approaches that have recently been used in uveal melanoma analysis. However, the presence of precursor clones and immune infiltrate in uveal melanoma have been described as being part of the tumour already decades ago. Since uveal melanoma grow in the corpus vitreous, they present a unique tumour model because every cell present in the tumour tissue is actually part of the tumour and possibly plays a role. For an effective treatment of uveal melanoma metastasis, it should be clear whether precursor clones and normal cells play an active role in progression and metastasis. We propagate analysis of bulk tissue that allows analysis of tumour heterogeneity in a clinical setting. Show less
Purpose: This study was designed to identify mitochondrial (mt) DNA variations in primary and metastatic uveal melanoma (UM) cell lines and their relation with cell metabolism to gain insight into... Show morePurpose: This study was designed to identify mitochondrial (mt) DNA variations in primary and metastatic uveal melanoma (UM) cell lines and their relation with cell metabolism to gain insight into metastatic progression.Method: The entire mtDNA genomes were sequenced using Sanger sequencing from two primary UM cell lines (92.1 and MEL270) and two cell lines (OMM2.3 and OMM2.5) derived from liver metastases of the MEL270 patient. The mtDNA copy numbers determined by the ratio of nDNA versus mtDNA. qRT-PCR was used to evaluate expression levels of mitochondrial biogenesis genes.Results: Sequencing showed that cell line MEL270 and metastases-derived OMM2.3 and OMM2.5 cell lines had homoplasmic single nucleotide polymorphisms (SNPs) representing J1c7a haplogroup, whereas 92.1 cells had mtDNA H31a haplogroup. mtDNA copy numbers were significantly higher in primary cell lines. The metastatic UM cells showed down-regulation of POLG, TFAM, NRF-1 and SIRT1 compared to their primary MEL270 cells. PGC-1 alpha was downregulated in 92.1 and upregulated in MEL270, OMM2.3 and OMM2.5.Conclusions: Our finding suggests that within metastatic cells, the heteroplasmic SNPs, copy numbers and mitochondrial biogenesis genes are modulated differentially compared to their primary UM cells. Therefore, investigating pathogenic mtDNA variants associated with cancer metabolic susceptibility may provide future therapeutic strategies in metastatic UM. (C) 2021 Published by Elsevier Inc. Show less
BackgroundTo understand how to improve the effect of immune checkpoint inhibitors in uveal melanoma (UM), we need a better understanding of the expression of PD-1 and PD-L1, their relation with the... Show moreBackgroundTo understand how to improve the effect of immune checkpoint inhibitors in uveal melanoma (UM), we need a better understanding of the expression of PD-1 and PD-L1, their relation with the presence of tumor-infiltrating lymphocytes (TILs), and their prognostic relevance in UM patients. Materials and methodsExpression of PD-1 and PD-L1 was assessed in 71 UM tissue samples by immunohistochemistry and quantitative real-time PCR (qRT-PCR), and further validated by western blotting. The effect of interferon gamma (IFN-gamma) on PD-1/PD-L1 expression was determined on four UM cell lines. ResultsImmunoreactivity of PD-1 was found in 30/71 cases and of PD-L1 in 44/71 UM samples. Tumor-infiltrating lymphocytes were found in 46% of UM tissues. PD-1 was expressed on TILs while tumor cells expressed PD-L1. UM with and without TILs showed expression of PD-1 in 69% and 18% cases, respectively (p=0.001). Similarly, PD-L1 was found in 75% of UM with TILs and in 50% of cases without TILs, respectively (p=0.03). DFS rate were lower in patients with TILs with expression of PD-1 and PD-L1, but the rate of DFS was higher with expression of PD-L1 in patients without TILs. After treatment of UM cell lines with IFN-gamma, PD-1 expression was induced in all UM cell lines whereas PD-L1 expression was found at a lower level in untreated cells, while expression also increased following treatment with IFN-gamma .ConclusionOur study suggests that increased infiltration with TILs promotes the aggressive behavior and suppresses the immune response of UM cells, thereby inhibiting immunotherapy. Show less
Zaks, O.; Gaber, D.; Ben-Yaakov, K.; Sharvit-Bader, M.; Goz, A.; Rotfogel, Z.; ... ; Aharoni-Simon, M. 2021
Uveal melanoma (UM) and conjunctival melanoma (CM) are ocular malignancies that give rise to life-threatening metastases. Although local disease can often be treated successfully, it is often... Show moreUveal melanoma (UM) and conjunctival melanoma (CM) are ocular malignancies that give rise to life-threatening metastases. Although local disease can often be treated successfully, it is often associated with significant vision impairment and treatments are often not effective against metastatic disease. Novel treatment modalities that preserve vision may enable elimination of small tumors and may prevent subsequent metastatic spread.Very few mouse models of metastatic CM and UM are available for research and for development of novel therapies. One of the challenges is to follow tumor growth in-vivo and to determine the right size for treatment, mainly of the posterior, choroidal melanoma. Hence, the purpose of this study was to establish a simple, noninvasive imaging tool that will simplify visualization and tumor follow-up in mouse models of CM and UM.Tumors were induced by inoculation of murine B16LS9 cells into the sub-conjunctival or the choroidal space of a C57BL/6 mouse eye under a surgical microscope. Five to ten days following injection, tumor size was assessed by Phoenix MicronIVTM image-guided Optical Coherence Tomography (OCT) imaging, which included a realtime camera view and OCT scan of the conjunctiva and the retina. In addition, tumor size was evaluated by ultrasound and histopathological examination of eye sections.Tumor growth was observed 5-9 days following sub-conjunctival or sub-retinal injection of seven-thousand or seventy-thousand cells, respectively. A clear tumor mass was detected at these regions using the MicronIVTM imaging system camera and OCT scans. Histology of eye sections confirmed the presence of tumor tissue. OCT allowed an accurate measurement of tumor size in the UM model and a qualitative assessment of tumor size in the CM model. Moreover, OCT enabled assessing the success rate of the choroidal tumor induction and importantly, predicted final tumor size already on the day of cell inoculation.In conclusion, by using a simple, non-invasive imaging tool, we were able to follow intraocular tumor growth of both CM and UM, and to define, already at the time of cell inoculation, a grading scale to evaluate tumor size. This tool may be utilized for evaluation of new mouse models for CM and UM, as well as for testing new therapies for these diseases. Show less
