PURPOSE. Although fundus photography is extensively used in ophthalmology, refraction prevents accurate distance measurement on fundus images, as the resulting scaling differs between subjects due... Show morePURPOSE. Although fundus photography is extensively used in ophthalmology, refraction prevents accurate distance measurement on fundus images, as the resulting scaling differs between subjects due to varying ocular anatomy. We propose a PARaxial Optical fundus Scaling (PAROS) method to correct for this variation using commonly available clinical data. METHODS. The complete optics of the eye and fundus camera were modeled using ray transfer matrix formalism to obtain fundus image magnification. The subject’s ocular geometry was personalized using biometry, spherical equivalent of refraction (RSE), keratometry, and/or corneal topography data. The PAROS method was validated using 41 different eye phantoms and subsequently evaluated in 44 healthy phakic subjects (of whom 11 had phakic intraocular lenses [pIOLs]), 29 pseudophakic subjects, and 21 patients with uveal melanoma. RESULTS. Validation of the PAROS method showed small differences between model and actual image magnification (maximum 3.3%). Relative to the average eye, large differences in fundus magnification were observed, ranging from 0.79 to 1.48. Magnification was strongly inversely related to RSE (R2 = 0.67). In phakic subjects, magnification was directly proportional to axial length (R2 = 0.34). The inverse relation was seen in pIOL (R2 = 0.79) and pseudophakic (R2 = 0.12) subjects. RSE was a strong contributor to magnification differences (1%–83%). As this effect is not considered in the commonly used Bennett–Littmann method, statistically significant differences up to 40% (mean absolute 9%) were observed compared to the PAROS method (P < 0.001). CONCLUSIONS. The significant differences in fundus image scaling observed among subjects can be accurately accounted for with the PAROS method, enabling more accurate quantitative assessment of fundus photography Show less
Background: Uveal melanoma (UM) is a rare intraocular tumor with a dismal prognosis once metastasized. This study provides a nationwide overview and time trends of patients diagnosed with primary... Show moreBackground: Uveal melanoma (UM) is a rare intraocular tumor with a dismal prognosis once metastasized. This study provides a nationwide overview and time trends of patients diagnosed with primary UM in the Netherlands between 1989 and 2019. Methods: A retrospective population-based cohort study based on patients with primary UM from the database of the Netherlands Cancer Registry (NCR), linked with the national population registry Statistics Netherlands on inhabitants' cause of death. Two time periods (1989-2004, 2005-2019) were compared with descriptive statistics. Kaplan-Meier and (multivariate) Cox proportional hazard models were used to assess changes over time for overall survival (OS) and cancer-specific survival (CSS). Results: In total, 5036 patients were analyzed with a median age of 64.0 years at the time of diagnosis. The number of patients increased over time. In the first (1989-2004) and second (2005-2019) period, 32% versus 54% of the patients received radiotherapy (p < 0.001). The median FU time was 13.4 years. The median OS of the first and second periods was 9.5 (95% CI 8.7-10.3) versus 11.3 years (95% CI 10.3-12.3; p < 0.001). The median CSS was 30.0 years (95% CI NA) in the first period and not reached in the second period (p = 0.008). In multivariate analysis (MVA), female gender (HR 0.85; 95% CI 0.79-0.92, p < 0.001) and radiotherapy treatment (HR 0.73; 95% CI 0.64-0.83, p < 0.001) were associated with better OS. Radiotherapy treatment (HR 0.74; 95% CI 0.61-0.90, p = 0.002) was also associated with better CSS. The period of diagnosis was not associated with OS or CSS. Conclusions: In this study of patients with primary UM, there was a shift to the diagnosis of smaller tumors, possibly due to stage migration. There was also an increase in eye-preserving treatments over time. OS and CSS were modestly improved in the second time period; however, the time period was not associated with OS or CSS in multivariate analyses. Show less
Uveal melanoma is the most common intraocular tumor in adults, representing approximately 5% of all melanoma cases. Up to 50% of uveal melanoma patients develop metastases that are resistant to... Show moreUveal melanoma is the most common intraocular tumor in adults, representing approximately 5% of all melanoma cases. Up to 50% of uveal melanoma patients develop metastases that are resistant to most of the commonly used antineoplastic treatments. Virtually all uveal melanoma tumors harbor activating mutations in GNAQ or GNA11, encoding Gαq and Gα11, respectively. Constant activity of these proteins causes deregulation of multiple downstream signaling pathways including PKC, MAPK and YAP1/TAZ. While the importance of YAP1 signaling for the proliferation of uveal melanoma has recently been demonstrated, much less is known about the paralog of YAP1 transcriptional coactivator, named TAZ; however, similar to YAP1, TAZ is expected to be a therapeutic target in uveal melanoma. We performed a small-scale drug screen to discover a compound synergistically inhibiting uveal melanoma proliferation/survival in combination with YAP1/TAZ inhibition. We found that the combination of genetic depletion of YAP1/TAZ together with Mcl-1 inhibition demonstrates a synergistic inhibitory effect on the viability of uveal melanoma cell lines. Similarly, indirect attenuation of the YAP1/TAZ signaling pathway with an inhibitor of the mevalonate pathway, that is, the geranyl-geranyl transferase inhibitor GGTI-298, synergizes with Mcl-1 inhibition. This combination could be potentially used as a treatment for metastatic uveal melanoma. Show less
BackgroundUveal melanoma is a disease characterized by constitutive activation of the G alpha pathway and downstream signaling of protein kinase C (PKC) and the mitogen-activated protein kinase ... Show moreBackgroundUveal melanoma is a disease characterized by constitutive activation of the G alpha pathway and downstream signaling of protein kinase C (PKC) and the mitogen-activated protein kinase (MAPK) pathway. While limited clinical activity has been observed in patients with metastatic disease with inhibition of PKC or MEK alone, preclinical data has demonstrated synergistic antitumor effects with concurrent inhibition of PKC and MEK. MethodWe conducted a phase Ib study of the PKC inhibitor sotrastaurin in combination with the MEK inhibitor binimetinib in patients with metastatic uveal melanoma using a Bayesian logistic regression model guided by the escalation with overdose control principle (NCT01801358). Serial blood samples and paired tumor samples were collected for pharmacokinetic (PK) and pharmacodynamic analysis. ResultsThirty-eight patients were treated across six dose levels. Eleven patients experienced DLTs across the five highest dose levels tested, most commonly including vomiting (n=3), diarrhea (n=3), nausea (n=2), fatigue (n=2) and rash (n=2). Common treatment related adverse events included diarrhea (94.7%), nausea (78.9%), vomiting (71.1%), fatigue (52.6%), rash (39.5%), and elevated blood creating phosphokinase (36.8%). Two dose combinations satisfying criteria for the maximum tolerated dose (MTD) were identified: (1) sotrastaurin 300 mg and binimetinib 30 mg; and, (2) sotrastaurin 200 mg and binimetinib 45 mg. Exposure to both drugs in combination was consistent with single-agent data for either drug, indicating no PK interaction between sotrastaurin and binimetinib. Stable disease was observed in 60.5% of patients treated. No patient achieved a radiographic response per RECIST v1.1. ConclusionsConcurrent administration of sotrastaurin and binimetinib is feasible but associated with substantial gastrointestinal toxicity. Given the limited clinical activity achieved with this regimen, accrual to the phase II portion of the trial was not initiated. Show less
Microphthalmia-associated transcription factor (MITF) is an important regulator of melanogenesis and melanocyte development. In cutaneous melanoma, MITF loss has been linked to an increased... Show moreMicrophthalmia-associated transcription factor (MITF) is an important regulator of melanogenesis and melanocyte development. In cutaneous melanoma, MITF loss has been linked to an increased expression of stem cell markers, a shift in epithelial-to-mesenchymal transition (EMT)-related factors, and increased inflammation. We explored the role of MITF in Uveal Melanoma (UM) using a cohort of 64 patients enucleated at the Leiden University Medical Center. We analysed the relation between MITF expression and clinical, histopathological and genetic features of UM, as well as survival. We performed differential gene expression and gene set enrichment analysis using mRNA microarray data, comparing MITF-low with MITF-high UM. MITF expression was lower in heavily pigmented UM than in lightly pigmented UM (p = 0.003), which we confirmed by immunohistochemistry. Furthermore, MITF was significantly lower in UM with monosomy 3/BAP1 loss than in those with disomy 3/no BAP1 loss (p < 0.001) and with 8q gain/amplification 8q (p = 0.02). Spearman correlation analysis showed that a low MITF expression was associated with an increase in inflammatory markers, hallmark pathways involved in inflammation, and epithelial-mesenchymal transition. Similar to the situation in cutaneous melanoma, we propose that MITF loss in UM is related to de-differentiation to a less favourable EMT profile and inflammation. Show less
Uveal melanoma (UM) is a rare malignant cancer of the eye, with up to 50% of patients dying from metastasis, for which no effective treatment is available. Due to the rarity of the disease, there... Show moreUveal melanoma (UM) is a rare malignant cancer of the eye, with up to 50% of patients dying from metastasis, for which no effective treatment is available. Due to the rarity of the disease, there is a great need to harness the limited material available from primary tumors and metastases for advanced research and preclinical drug screening. We established a platform to isolate, preserve, and transiently recover viable tissues, followed by the generation of spheroid cultures derived from primary UM. All assessed tumor-derived samples formed spheroids in culture within 24 h and stained positive for melanocyte-specific markers, indicating the retention of their melanocytic origin. These short-lived spheroids were only maintained for the duration of the experiment (7 days) or re-established from frozen tumor tissue acquired from the same patient. Intravenous injection of fluorescently labeled UM cells derived from these spheroids into zebrafish yielded a reproducible metastatic phenotype and recapitulated molecular features of the disseminating UM. This approach allowed for the experimental replications required for reliable drug screening (at least 2 individual biological experiments, with n > 20). Drug treatments with navitoclax and everolimus validated the zebrafish patient-derived model as a versatile preclinical tool for screening anti-UM drugs and as a preclinical platform to predict personalized drug responses. Show less
Simple Summary Uveal melanoma is a rare and aggressive disease. G alpha-proteins GNAQ and GNA11 are driver mutations that activate MAP kinase and YAP/TAZ pathways. BAP1 loss and monosomy of... Show moreSimple Summary Uveal melanoma is a rare and aggressive disease. G alpha-proteins GNAQ and GNA11 are driver mutations that activate MAP kinase and YAP/TAZ pathways. BAP1 loss and monosomy of chromosome 3 are present in patients with high risk of metastasis. MEK-inhibitors do not significantly block UM progression. Combinations of the MEK inhibitor trametinib and different classes of drugs targeting YAP/TAZ were used to overcome resistance. Combination of trametinib and cerivastatin were synergistic in vitro and in vivo in BAP1 mutated and chromosome 3 monosomic uveal melanoma cell lines. Background: Metastatic uveal melanoma (MUM) is a highly aggressive, therapy-resistant disease. Driver mutations in G alpha-proteins GNAQ and GNA11 activate MAP-kinase and YAP/TAZ pathways of oncogenic signalling. MAP-kinase and MEK-inhibitors do not significantly block MUM progression, likely due to persisting YAP/TAZ signalling. Statins inhibit YAP/TAZ activation by blocking the mevalonate pathway, geranyl-geranylation, and subcellular localisation of the Rho-GTPase. We investigated drugs that affect the YAP/TAZ pathway, valproic acid, verteporfin and statins, in combination with MEK-inhibitor trametinib. Methods: We established IC50 values of the individual drugs and monitored the effects of their combinations in terms of proliferation. We selected trametinib and cerivastatin for evaluation of cell cycle and apoptosis. Synergism was detected using isobologram and Chou-Talalay analyses. The most synergistic combination was tested in vivo. Results: Synergistic concentrations of trametinib and cerivastatin induced a massive arrest of proliferation and cell cycle and enhanced apoptosis, particularly in the monosomic, BAP1-mutated UPMM3 cell line. The combined treatment reduced ERK and AKT phosphorylation, increased the inactive, cytoplasmatic form of YAP and significantly impaired the growth of UM cells with monosomy of chromosome 3 in NSG mice. Conclusion: Statins can potentiate the efficacy of MEK inhibitors in the therapy of UM. Show less
PURPOSE. Uveal melanoma (UM) is considered a rare disease; yet, it is the most common intraocular malignancy in adults. Although the primary tumor may be efficiently managed, more than 50% of... Show morePURPOSE. Uveal melanoma (UM) is considered a rare disease; yet, it is the most common intraocular malignancy in adults. Although the primary tumor may be efficiently managed, more than 50% of patients with UM develop distant metastases. The mortality at the first year after diagnosis of metastatic UM has been estimated at 81%, and the poor prognosis has not improved in the past years due to the lack of effective therapies. METHODS. In order to search for novel therapeutic possibilities for metastatic UM, we performed a small-scale screen of targeted drug combinations. We verified the targets of the tested compounds by western blotting and PCR and clarified the mechanism of action of the selected combinations by caspase 3 and 7 activity assay and flow cytometry. The best two combinations were tested in a mouse patient-derived xenograft (PDX) UM model as putative therapeutics for metastatic UM. RESULTS. Combinations of the multitarget drug trabectedin with either the CK2/CLK double-inhibitor CX-4945 (silmitasertib) or the c-MET/TAM (TYRO3, Axl, MERTK) receptor inhibitors foretinib and cabozantinib demonstrated synergistic effects and induced apoptosis (relative caspase 3 and 7 activity increased up to 20.5-fold in UM cell lines). In the case of the combination of foretinib and cabozantinib, inhibition of the TAM receptors, but not c-Met, was essential to inhibit the growth of UM cells. Monotreatment with trabectedin inhibited tumor growth by 42%, 49%, and 35% in the MM26, MM309, and MM339 PDX mouse models, respectively. CONCLUSIONS. Trabectedin alone or in combination with cabozantinib inhibited tumor growth in PDX UM mouse models. Blocking of MERTK, rather than TYRO3, activity inhibited UM cell growth and synergized with trabectedin. Show less
The tumor suppressor protein p53 has an important role in cell-fate determination. In cancer cells, the activity of p53 is frequently repressed by high levels of MDMX and/or MDM2. MDM2 is a... Show moreThe tumor suppressor protein p53 has an important role in cell-fate determination. In cancer cells, the activity of p53 is frequently repressed by high levels of MDMX and/or MDM2. MDM2 is a ubiquitin ligase whose activity results in ubiquitin- and proteasome-dependent p53 degradation, while MDMX inhibits p53-activated transcription by shielding the p53 transactivation domain. Interestingly, the oncogenic functions of MDMX appear to be more wide-spread than inhibition of p53. The present study aimed to elucidate the MDMX-controlled transcriptome. Therefore, we depleted MDMX with four distinct shRNAs from a high MDMX expressing uveal melanoma cell line and determined the effect on the transcriptome by RNAseq. Biological function analyses indicate the inhibition of the cell cycle regulatory genes and stimulation of cell death activating genes upon MDMX depletion. Although the inhibition of p53 activity clearly contributes to the transcription regulation controlled by MDMX, it appeared that the transcriptional regulation of multiple genes did not only rely on p53 expression. Analysis of gene regulatory networks indicated a role for Forkhead box (FOX) transcription factors. Depletion of FOXO proteins partly prevented the transcriptional changes upon MDMX depletion. Furthermore, depletion of FOXO proteins relatively diminished the growth inhibition upon MDMX knockdown, although the knockdown of the FOXO transcription factors also reduces cell growth. In conclusion, the p53-independent oncogenic functions of MDMX could be partially explained by its regulation of FOXO activity. Show less
Simple Summary: We performed a CRISPR-Cas9 synthetic lethality screen in order to identify molecular targets whose inhibition would synergistically enhance the effect of everolimus in uveal... Show moreSimple Summary: We performed a CRISPR-Cas9 synthetic lethality screen in order to identify molecular targets whose inhibition would synergistically enhance the effect of everolimus in uveal melanoma cells. IGF1R and PRKDC, among others, were identified as hits. We verified these hits effects genetically: we treated the uveal melanoma cell lines depleted of PRKDC or IGF1R with everolimus and, in case of IGF1R, observed a synergistic effect. Additionally, we found synergistic growth inhibition with the inhibitors targeting DNA-PKcs or IGF1R in combination with everolimus. Moreover, we investigated the combination of targeted inhibitors of DNA-PKcs and IGF1R with everolimus on uveal melanoma in an in vivo model. The dual DNA-PKcs/mTOR inhibitor CC-115 demonstrated activity in vivo. Currently, no systemic treatment is approved as the standard of care for metastatic uveal melanoma (UM). mTOR has been evaluated as a drug target in UM. However, one of the main limitations is dose reduction due to adverse effects. The combination of everolimus with another targeted agent would allow the reduction of the dose of a single drug, thus widening the therapeutic window. In our study, we aimed to identify a synergistic combination with everolimus in order to develop a novel treatment option for metastatic UM. We exploited CRISPR-Cas9 synthetic lethality screening technology to search for an efficient combination. IGF1R and PRKDC and several other genes were identified as hits in the screen. We investigated the effect of the combination of everolimus with the inhibitors targeting IGF1R and DNA-PKcs on the survival of UM cell lines. These combinations synergistically slowed down cell growth but did not induce apoptosis in UM cell lines. These combinations were tested on PDX UM in an in vivo model, but we could not detect tumor regression. However, we could find significant activity of the dual DNA-PKcs/mTOR inhibitor CC-115 on PDX UM in the in vivo model. Show less
Microphthalmia-associated transcription factor (MITF) is an important regulator of melanogenesis and melanocyte development. Although it has been studied extensively in cutaneous melanoma, the role... Show moreMicrophthalmia-associated transcription factor (MITF) is an important regulator of melanogenesis and melanocyte development. Although it has been studied extensively in cutaneous melanoma, the role of MITF in uveal melanoma (UM) has not been explored in much detail. We review the literature about the role of MITF in normal melanocytes, in cutaneous melanoma, and in UM. In normal melanocytes, MITF regulates melanocyte development, melanin synthesis, and melanocyte survival. The expression profile and the behaviour of MITF-expressing cells suggest that MITF promotes local proliferation and inhibits invasion, inflammation, and epithelial-to-mesenchymal (EMT) transition. Loss of MITF expression leads to increased invasion and inflammation and is more prevalent in malignant cells. Cutaneous melanoma cells switch between MITF-high and MITF-low states in different phases of tumour development. In UM, MITF loss is associated with loss of BAP1 protein expression, which is a marker of poor prognosis. These data indicate a dual role for MITF in benign and malignant melanocytic cells. Show less
Marinkovic, M.; Pors, L.J.; Berg, V. van den; Peters, F.P.; Schalenbourg, A.; Zografos, L.; ... ; Horeweg, N. 2021
Simple Summary This study aims to assess cancer control and preservation of the eye and visual acuity after proton therapy abroad for eye melanoma. For this, medical files were reviewed of Dutch... Show moreSimple Summary This study aims to assess cancer control and preservation of the eye and visual acuity after proton therapy abroad for eye melanoma. For this, medical files were reviewed of Dutch uveal melanoma patients who were treated in Switzerland with proton therapy from 1987 to 2019. The tumours of these patients were too large and/or localised too close to the optic nerve to be treated with local plaque irradiation. There were 103 patients, of whom one had a uveal melanoma in both eyes. The tumours were relatively large and often localised around the central part of the retina. At five years after treatment, proton therapy had controlled the uveal melanomas of 94% of the patients and 81% had preserved their eye. Spread of the cancer beyond the eye was observed in 30% of the patients. Most patients (79%) became blind or had severe visual impairment after proton therapy; a small group of patients had mild or no visual impairment (17%). The size of the tumour, its localisation and the dose of proton therapy were important for the risk of decline in visual acuity. This study shows that proton therapy abroad for uveal melanoma is feasible and yields good results. Objective: To assess oncological and ophthalmological outcomes after international referral of uveal melanoma patients for proton therapy. Materials and Methods: This is a retrospective study among Dutch uveal melanoma patients who were treated in Switzerland with 60.0 CGE proton therapy (in 4 fractions) from 1987 to 2019. All patients were ineligible for brachytherapy due to tumour size and/or proximity to the optic nerve. Time-to-event analyses were performed using Kaplan-Meier's methodology and Cox proportional hazards models. Results: There were 103 patients (104 eyes) with a median largest tumour diameter of 19 mm (range 6-26 mm). Tumours were localised centrally (11%), mid-peripherally (65%) or peripherally (34%). Median follow-up was 7 years. Five-year local control, distant metastasis-free survival and eye preservation rates were 94%, 70% and 81% respectively. At five years, severe, moderate and mild visual impairment was observed in respectively 79%, 4% and 6% of the patients. Larger tumour volumes and more central tumour localisation were associated with severe visual impairment. After correction for these factors, dose to the macula, optic disc and retina, but not optic nerve was significantly associated with severe visual impairment. Conclusion: International referral for proton therapy yielded good tumour control and eye preservation rates, but risk of distant metastasis and severe visual impairment were substantial, possibly due to the selection of advanced tumour stages and/or central localisation. Dose to the macula may be more relevant than dose to the optic nerve for preservation of visual acuity, which is relevant for the treatment planning of proton therapy. Show less
Background Magnetic resonance imaging (MRI) is increasingly being used in the diagnosis and treatment planning of uveal melanoma (UM), the most common primary intraocular tumor. Initially, 7 T MRI... Show moreBackground Magnetic resonance imaging (MRI) is increasingly being used in the diagnosis and treatment planning of uveal melanoma (UM), the most common primary intraocular tumor. Initially, 7 T MRI was primarily used, but more recently these techniques have been translated to 3 T, as it is more commonly available. Purpose Compare the diagnostic performance of 3 T and 7 T MRI of UM. Study Type Prospective. Population Twenty-seven UM patients (19% female). Field Strength/Sequence 3 T: T1- and T2-weighted three-dimensional (3D) spin echo (SE) and multi-slice (MS) SE, 7 T: T1-weighted 3D gradient echo (GE), T2-weighted 3D SE and MS SE, 3 T and 7 T GE dynamic contrast-enhanced. T1 weighted images: acquired before and after Gadolinium (Gd) administration. Assessment For all sequences, scan and diagnostic quality was quantified using a 5-point Likert scale. Signal intensities on T1 and T2 relative to choroid and eye muscle respectively were assessed as well as the tumor prominence. Finally, the perfusion time-intensity curves (TICs) were classified as plateau, progressive, or wash-out. Statistical Tests Image quality scores were compared between both field strengths using Wilcoxon signed-rank and McNemar tests. Paired t-tests and Bland-Altman were used for comparing tumor prominences. P < 0.05 was considered statistically significant. Results Image quality was comparable between 3 T and 7 T, for 3DT1, 3DT2, 3DT1Gd (P = 0.86; P = 0.34; P = 0.78, respectively) and measuring tumor dimensions (P = 0.40). 2DT1 and 2DT2 image quality were rated better on 3 T compared to 7 T. Most UM had the same relative signal intensities at 3 T and 7 T on T1 (17/21) and T2 (13/17), and 16/18 diagnostic TICs received the same classification. Tumor prominence measurements were similar between field strengths (95% confidence interval: -0.37 mm to 0.03 mm, P = 0.097). Data Conclusion Diagnostic performance of the evaluated 3 T protocol proved to be as capable as 7 T, with the addition of 3 T being superior in assessing tumor growth into nearby anatomical structures compared to 7 T. Level of Evidence 2 Technical Efficacy Stage 3 Show less
Souri, Z.; Wierenga, A.P.A.; Kroes, W.G.M.; Velden, P.A. van der; Verdijk, R.M.; Eikmans, M.; ... ; Jager, M.J. 2021
Simple Summary Uveal melanoma (UM) is a rare type of intraocular malignancy, which often gives rise to metastases. While treatment with immune checkpoint inhibitors is often effective in the... Show moreSimple Summary Uveal melanoma (UM) is a rare type of intraocular malignancy, which often gives rise to metastases. While treatment with immune checkpoint inhibitors is often effective in the treatment of cutaneous melanoma metastases, it is hardly effective in the case of UM metastases. Lymphocyte-activation gene 3 (LAG3) is a recently recognized immune checkpoint; we determined the distribution of LAG3 expression and its ligands in three sets of primary UM. High-risk UM (epithelioid cell type, loss of chromosome 3/BAP1 staining) had a higher expression of LAG3 and its ligands, which correlated with the presence of infiltrating immune cells. We concluded that not only LAG3 but also its ligands Galectin-3 and HLA class II are especially expressed in high-risk UM and may be a target for adjuvant immunotherapy in UM. Uveal melanoma (UM) is a rare ocular malignancy which originates in the uveal tract, and often gives rise to metastases. Potential targets for immune checkpoint inhibition are lymphocyte-activation gene 3 (LAG3) and its ligands. We set out to analyse the distribution of these molecules in UM. The expression of mRNA was determined using an Illumina array in 64 primary UM from Leiden. The T lymphocyte fraction was determined by digital droplet PCR. In a second cohort of 15 cases from Leiden, mRNA expression was studied by Fluidigm qPCR, while a third cohort consisted of 80 UM from TCGA. In the first Leiden cohort, LAG3 expression was associated with the presence of epithelioid cells (p = 0.002), monosomy of chromosome 3 (p = 0.004), and loss of BAP1 staining (p = 0.001). In this Leiden cohort as well as in the TCGA cohort, LAG3 expression correlated positively with the expression of its ligands: LSECtin, Galectin-3, and the HLA class II molecules HLA-DR, HLA-DQ, and HLA-DP (all p < 0.001). Furthermore, ligands Galectin-3 and HLA class II were increased in monosomy 3 tumours and the expression of LAG3 correlated with the presence of an inflammatory phenotype (T cell fraction, macrophages, HLA-A and HLA-B expression: all p < 0.001). High expression levels of LAG3 (p = 0.01), Galectin-3 (p = 0.001), HLA-DRA1 (p = 0.002), HLA-DQA1 (p = 0.04), HLA-DQB2 (p = 0.03), and HLA-DPA1 (p = 0.007) were associated with bad survival. We conclude that expression of the LAG ligands Galectin-3 and HLA class II strongly correlates with LAG3 expression and all are increased in UM with Monosomy 3/BAP1 loss. The distribution suggests a potential benefit of monoclonal antibodies against LAG3 or Galectin-3 as adjuvant treatment in patients with high-risk UM. Show less
Simple Summary We hypothesize that proangiogenic factors such as angiopoietin-1 (ANG-1) and angiopoietin-2 (ANG-2), two targetable cytokines, may play a role in tumor development in uveal melanoma.... Show moreSimple Summary We hypothesize that proangiogenic factors such as angiopoietin-1 (ANG-1) and angiopoietin-2 (ANG-2), two targetable cytokines, may play a role in tumor development in uveal melanoma. We determined the expression of these cytokines in both uveal melanoma tissue as well as in aqueous humor, which provides a unique combination of data. We observed that ANG-2, in contrast to ANG-1, showed more expression in high-risk tumors and was associated with the development of metastases. Its presence in aqueous humor correlated with expression in tumor tissue. Knowledge about the expression of these cytokines may help to identify targets for personalized treatment. Uveal melanoma (UM) metastasize haematogeneously, and tumor blood vessel density is an important prognostic factor. We hypothesized that proangiogenic factors such as angiopoietin-1 (ANG-1) and angiopoietin-2 (ANG-2), two targetable cytokines, might play a role in tumor development and metastatic behavior. mRNA levels of ANG-1 and ANG-2 were determined in 64 tumors using an Illumina HT-12 v4 mRNA chip and compared to clinical, pathologic, and genetic tumor parameters. Tissue expression was also determined by immunohistochemistry (IHC). Samples of aqueous humor were collected from 83 UM-containing enucleated eyes and protein levels that were determined in a multiplex proximity extension assay. High tissue gene expression of ANG-2, but not of ANG-1, was associated with high tumor thickness, high largest basal diameter, involvement of the ciliary body, and with UM-related death (ANG-2 mRNA p < 0.001; ANG-2 aqueous protein p < 0.001). The presence of the ANG-2 protein in aqueous humor correlated with its mRNA expression in the tumor (r = 0.309, p = 0.03). IHC showed that ANG-2 was expressed in macrophages as well as tumor cells. The presence of ANG-2 in the tumor and in aqueous humor, especially in high-risk tumors, make ANG-2 a potential targetable cytokine in uveal melanoma. Show less
Simple Summary Uveal melanoma (UM) is an ocular malignancy which is derived from melanocytes in the uveal tract. Epigenetic regulators such as Histone Deacetylase (HDACs) inhibitors are being... Show moreSimple Summary Uveal melanoma (UM) is an ocular malignancy which is derived from melanocytes in the uveal tract. Epigenetic regulators such as Histone Deacetylase (HDACs) inhibitors are being tested as treatment of UM metastases. Expression of different HDACs is variable, and some are increased in high-risk tumors with loss of one chromosome 3. As this genetic abnormality is also associated with an inflammatory phenotype, we analyzed whether HDAC expression was influenced by inflammation. In two cohorts of UM cases, expression of several HDACs showed a positive correlation with tumor-infiltrating T cells, while HDACs 2 and 11 showed a negative association with macrophages. Interferon-gamma stimulated expression of some HDACs on UM cell lines. These data suggest that cytokines produced by T cells may be responsible for the increased expression of some HDACs in UM with monosomy 3. In Uveal Melanoma (UM), an inflammatory phenotype is strongly associated with the development of metastases and with chromosome 3/BAP1 expression loss. As an increased expression of several Histone Deacetylases (HDACs) was associated with loss of chromosome 3, this suggested that HDAC expression might also be related to inflammation. We analyzed HDAC expression and the presence of leukocytes by mRNA expression in two sets of UM (Leiden and TCGA) and determined the T lymphocyte fraction through ddPCR. Four UM cell lines were treated with IFN gamma (50IU, 200IU). Quantitative PCR (qPCR) was used for mRNA measurement of HDACs in cultured cells. In both cohorts (Leiden and TCGA), a positive correlation occurred between expression of HDACs 1, 3 and 8 and the presence of a T-cell infiltrate, while expression of HDACs 2 and 11 was negatively correlated with the presence of tumor-infiltrating macrophages. Stimulation of UM cell lines with IFN gamma induced an increase in HDACs 1, 4, 5, 7 and 8 in two out of four UM cell lines. We conclude that the observed positive correlations between HDAC expression and chromosome 3/BAP1 loss may be related to the presence of infiltrating T cells. Show less
Simple Summary Uveal melanoma (UM) is a rare ocular malignancy that often gives rise to metastases. Tumours with an inflammatory phenotype have an especially bad prognosis. As an increased HLA... Show moreSimple Summary Uveal melanoma (UM) is a rare ocular malignancy that often gives rise to metastases. Tumours with an inflammatory phenotype have an especially bad prognosis. As an increased HLA expression and the presence of tumour-infiltrating lymphocytes and macrophages may be regulated by miRNAs, we set out to investigate whether any miRNAs are associated with inflammatory parameters in this malignancy. Some miRNAs were increased in UM with a high HLA expression and high T cell numbers, while others were decreased, showing two opposing patterns; however, both patterns were related to the tumour's chromosome 3/BAP1 status. We conclude that specific miRNAs are related to the inflammatory phenotype and that these are differentially expressed between disomy 3/BAP1-positive versus monosomy 3/BAP1-negative UM. MicroRNAs are known to play a role in the regulation of inflammation. As a high HLA Class I expression is associated with a bad prognosis in UM, we set out to determine whether any miRNAs were related to a high HLA Class I expression and inflammation. We also determined whether such miRNAs were related to the UM's genetic status. The expression of 125 miRNAs was determined in 64 primary UM from Leiden. Similarly, the mRNA expression of HLA-A, HLA-B, TAP1, BAP1, and immune cell markers was obtained. Expression levels of 24 of the 125 miRNAs correlated with expression of at least three out of four HLA Class I probes. Four miRNAs showed a positive correlation with HLA expression and infiltration with leukocytes, 20 a negative pattern. In the first group, high miRNA levels correlated with chromosome 3 loss/reduced BAP1 mRNA expression, in the second group low miRNA levels. The positive associations between miRNA-22 and miRNA-155 with HLA Class I were confirmed in the TCGA study and Rotterdam cohort, and with TAP1 in the Rotterdam data set; the negative associations between miRNA-125b2 and miRNA-211 and HLA-A, TAP1, and CD4 were confirmed in the Rotterdam set. We demonstrate two patterns: miRNAs can either be related to a high or a low HLA Class I/TAP1 expression and the presence of infiltrating lymphocytes and macrophages. However, both patterns were associated with chromosome 3/BAP1 status, which suggests a role for BAP1 loss in the regulation of HLA expression and inflammation in UM through miRNAs. Show less
Brouwer, N.J.; Konstantinou, E.K.; Gragoudas, E.S.; Marinkovic, M.; Luyten, G.P.M.; Kim, I.K.; ... ; Vavvas, D.G. 2021
PURPOSE. The purpose of this study was to determine whether YAP/TAZ activation in uveal melanoma (UM) and the susceptibility of melanoma cell lines to YAP/TAZ inhibition by verteporfin (VP) is... Show morePURPOSE. The purpose of this study was to determine whether YAP/TAZ activation in uveal melanoma (UM) and the susceptibility of melanoma cell lines to YAP/TAZ inhibition by verteporfin (VP) is related to the tumor's genetic background.METHODS. Characteristics of 144 patients with enucleated UM were analyzed together with mRNA expression levels of YAP/TAZ-related genes (80 patients from the The Cancer Genome Atlas [TCGA] project and 64 patients from Leiden, The Netherlands). VP was administered to cell lines 92.1, OMM1, Mel270, XMP46, and MM28 (UM), CRMM1 and CRMM2 (conjunctival melanoma), and OCM3 (cutaneous melanoma). Viability, growth speed, and expression of YAP1-related proteins were assessed.RESULTS. In TCGA data, high expression of YAP1 and WWTR1 correlated with the presence of monosomy 3 (P = 0.009 and P < 0.001, respectively) and BAP1-loss (P = 0.003 and P = 0.001, respectively) in the primary UM; metastasis development correlated with higher expression of YAP1 (P = 0.05) and WWTR1 (P = 0.003). In Leiden data, downstream transcription factor TEAD4 was increased in cases with M3/BAP1-loss (P = 0.002 and P = 0.006) and related to metastasis (P = 0.004). UM cell lines 92.1, OMM1, and Mel270 (GNAQ/11-mutation, BAP1-positive) and the fast-growing cell line OCM3 (BRAF-mutation) showed decreased proliferation after exposure to VP. Two slow-growing UM cell lines XMP46 and MM28 (GNAQ/11-mutation, BAP1-negative) were not sensitive to VP, and neither were the two conjunctival melanoma cell lines (BRAF/NRAS-mutation).CONCLUSIONS. High risk UM showed an increased expression of YAP/TAZ-related genes. Although most UM cell lines responded in vitro to VP, BAP1-negative and conjunctival melanoma cell lines did not. Not only the mutational background, but also cell growth rate is an important predictor of response to YAP/TAZ inhibition by VP. Show less
Fleury, E.; Trnkova, P.; Erdal, E.; Hassan, M.; Stoel, B.; Jaarma-Coes, M.; ... ; Hoogeman, M. 2021
Purpose: To develop a high-resolution three-dimensional (3D) magnetic resonance imaging (MRI)-based treatment planning approach for uveal melanomas (UM) in proton therapy.Materials/methods: For... Show morePurpose: To develop a high-resolution three-dimensional (3D) magnetic resonance imaging (MRI)-based treatment planning approach for uveal melanomas (UM) in proton therapy.Materials/methods: For eight patients with UM, a segmentation of the gross tumor volume (GTV) and organs-at-risk (OARs) was performed on T1- and T2-weighted 7 Tesla MRI image data to reconstruct the patient MR-eye. An extended contour was defined with a 2.5-mm isotropic margin derived from the GTV. A broad beam algorithm, which we have called pi Dose, was implemented to calculate relative proton absorbed doses to the ipsilateral OARs. Clinically favorable gazing angles of the treated eye were assessed by calculating a global weighted-sum objective function, which set penalties for OARs and extreme gazing angles. An optimizer, which we have named OPT'im-Eye-Tool, was developed to tune the parameters of the functions for sparing critical-OARs.Results: In total, 441 gazing angles were simulated for every patient. Target coverage including margins was achieved in all the cases (V-95% > 95%). Over the whole gazing angles solutions space, maximum dose (D-max) to the optic nerve and the macula, and mean doses (D-mean) to the lens, the ciliary body and the sclera were calculated. A forward optimization was applied by OPT'im-Eye-Tool in three different prioritizations: iso-weighted, optic nerve prioritized, and macula prioritized. In each, the function values were depicted in a selection tool to select the optimal gazing angle(s). For example, patient 4 had a T2 equatorial tumor. The optimization applied for the straight gazing angle resulted in objective function values of 0.46 (iso-weighted situation), 0.90 (optic nerve prioritization) and 0.08 (macula prioritization) demonstrating the impact of that angle in different clinical approaches.Conclusions: The feasibility and suitability of a 3D MRI-based treatment planning approach have been successfully tested on a cohort of eight patients diagnosed with UM. Moreover, a gaze-angle trade-off dose optimization with respect to OARs sparing has been developed. Further validation of the whole treatment process is the next step in the goal to achieve both a non-invasive and a personalized proton therapy treatment. (c) 2020 The Authors. Medical Physics published by Wiley Periodicals LLC on behalf of American Association of Physicists in Medicine. Show less
Souri, Z.; Jochemsen, A.G.; Versluis, M.; Wierenga, A.P.A.; Nemati, F.; Velden, P.A. van der; ... ; Jager, M.J. 2020
Simple SummaryChemotherapy and immunotherapy are both used to treat malignancies. The immunotherapy of cancer often involves T cells, which recognise the antigens presented in HLA molecules. Uveal... Show moreSimple SummaryChemotherapy and immunotherapy are both used to treat malignancies. The immunotherapy of cancer often involves T cells, which recognise the antigens presented in HLA molecules. Uveal melanoma (UM) is an intraocular malignancy, which often gives rise to metastases. We determined whether high-risk tumours expressed the target of two drugs, histone deacetylase (HDAC) inhibitor Quisinostat and Tazemetostat, an inhibitor of Enhancer of zeste homologue 2 (EZH2). We observed that especially high-risk UM tumours (monosomy 3, gain of 8q, loss of BAP1) expressed several HDACs, and showed a high HLA Class I expression. We further tested whether these drugs influenced HLA Class I expression on three UM cell lines. The drug Quisinostat led to an upregulation of HLA protein and mRNA levels in three UM cell lines, while Tazemetostat had little effect. We concluded that the use of drugs that influence epigenetic regulators may impact immunotherapy approaches.The treatment of uveal melanoma (UM) metastases or adjuvant treatment may imply immunological approaches or chemotherapy. It is to date unknown how epigenetic modifiers affect the expression of immunologically relevant targets, such as the HLA Class I antigens, in UM. We investigated the expression of HDACs and the histone methyl transferase EZH2 in a set of 64 UMs, using an Illumina HT12V4 array, and determined whether a histone deacetylase (HDAC) inhibitor and EZH2 inhibitor modified the expression of HLA Class I on three UM cell lines. Several HDACs (HDAC1, HDAC3, HDAC4, and HDAC8) showed an increased expression in high-risk UM, and were correlated with an increased HLA expression. HDAC11 had the opposite expression pattern. While in vitro tests showed that Tazemetostat did not influence cell growth, Quisinostat decreased cell survival. In the three tested cell lines, Quisinostat increased HLA Class I expression at the protein and mRNA level, while Tazemetostat did not have an effect on the cell surface HLA Class I levels. Combination therapy mostly followed the Quisinostat results. Our findings indicate that epigenetic drugs (in this case an HDAC inhibitor) may influence the expression of immunologically relevant cell surface molecules in UM, demonstrating that these drugs potentially influence immunotherapy. Show less
