Search

Purpose: Cell lines are being used in preclinical uveal melanoma (UM) research. Because not all cell lines harbor typical GNAQ or GNA11 hotspot mutations, we aimed at better classifying them and determining whether we could find genetic causes to explain the protein and mRNA expression profiles of the cell lines. Methods: We studied protein and mRNA expression of 14 UM cell lines and determined the presence of single nucleotide variants and small insertions and deletions with next-generation sequencing and copy number alterations with a single nucleotide polymorphism array. The lists of differentially expressed proteins and genes were merged, and shared lists were created, keeping only terms with concordant mRNA and protein expression. Enrichment analyses were performed on the shared lists. Results:Cell lines Mel285 and Mel290 are separate from GNA-mutated cell lines and show downregulation of melanosome-related markers. Both lack typical UM mutations but each harbors four putatively deleterious variants in CTNNB1, PPP1R10, LIMCH1, and APC in Mel285 and ARID1A, PPP1R10, SPG11, and RNF43 in Mel290. The upregulated terms in Mel285 and Mel290 did not point to a convincing alternative origin. Mel285 shows loss of chromosomes 1p, 3p, partial 3q, 6, and partial 8p, whereas Mel290 shows loss of 1p and 6. Expression in the other 12 cell lines was related to BAP1 expression.Conclusions: Although Mel285 and Mel290 have copy number alterations that fit UM, multi-omics analyses show that they belong to a separate group compared to the other analyzed UM cell lines. Therefore, they may not be representative models to test potential therapeutic targets for UM.

Spinocerebellar Ataxia Type 7 (SCA7) is an autosomal dominantly inherited disorder, primarily characterized by cerebellar ataxia and visual loss. SCA7 is caused by a CAG repeat expansion in exon 3 of the ATXN7 gene. We generated human induced pluripotent stem cells (hiPSCs) from peripheral blood-derived erythroblasts from two SCA7 patients (LUMCi051-A,B and LUMCi052-A,B,C) using integration-free episomal vectors. All hiPSC clones express pluripotency factors, show a normal karyotype, and can differentiate into the three germ layers. These lines can be used for in vitro disease modeling and therapy testing.

Purpose: Uveal melanoma (UM) is the most common primary intraocular malignancy with a high probability of metastatic disease. Although excellent treatment options for primary UM are available, therapy for metastatic disease remain limited. Drug discovery studies using mouse models have thus far failed to provide therapeutic solutions, highlighting the need for novel models. Here, we optimize zebrafish xenografts as a potential model for drug discovery by showcasing the behavior of multiple cell lines and novel findings on mutation-dependent compound synergism/antagonism using Z-Tada; an algorithm to objectively characterize output measurements. Methods: Prognostic relevant primary (N = 4) and metastatic UM (N = 1) cell lines or healthy melanocytes (N = 2) were inoculated at three distinct inoculation sites. Standardized quantifications independent of inoculation site were obtained using Z-Tada; an algorithm to measure tumor burden and the number, size, and distance of disseminated tumor cells. Sequentially, we utilized this model to validate combinatorial synergism or antagonism seen in vitro. Results: Detailed analysis of 691 zebrafish xenografts demonstrated perivitelline space inoculation provided robust data with high probability of cell dissemination. Cell lines with more invasive behavior (SF3B1mut and BAP1mut) behaved most aggressive in this model. Combinatorial drug treatment illustrated synergism or antagonism is mutation-dependent, which were confirmed in vivo. Combinatorial treatment differed per xenograft-model, as it either inhibited overall tumor burden or cell dissemination. Conclusions: Perivitelline space inoculation provides robust zebrafish xenografts with the ability for high-throughput drug screening and robust data acquisition using Z-Tada. This model demonstrates that drug discovery for uveal melanoma must take mutational subclasses into account, especially in comb

Purpose: Gain of chromosome 8q has been associated with poor prognosis in uveal melanoma (UM), and an increase in the absolute number of 8q-copies correlated with an even shorter survival. Splicing factor 3b subunit 1 (SF3B1)-mutated (SF3B1 MUT) tumors display structural chromosomal anomalies and frequently show a partial gain of chromosome 8qter. A recent subset of SF3B1 MUT UM with early-onset metastases has been identified, prompting the investigation of the relationship between survival, 8q gain, and SF3B1 MUT UM. Design: Retrospective cohort study. Subjects: Patients diagnosed with UM who underwent enucleation or received a biopsy at the Erasmus MC Cancer Institute or the Rotterdam Eye Hospital, The Netherlands were included. Methods: Fifty-nine patients with SF3B1 MUT tumors and 211 patients with BRCA1 associated protein 1 (BAP1)-mutated (BAP1 MUT) tumors were included in this study. Copy number status and gene expression were assessed using either a single nucleotide polymorphism array, fluorescence in situ hybridization, and karyotyping, or a combination of these techniques. Disease-free survival was determined and a cut-off of 60 months was used to define early-onset metastatic disease. Main Outcome Measures: Disease-free survival. Results: Forty-eight patients with SF3B1 MUT UM (81%) had chromosome 8q gain (3 copies, 78%; 4 copies, 22%). Kaplan–Meier analysis of SF3B1 MUT UM did not indicate a difference in survival in patients with or without gain of 8q (P = 0.99). Furthermore, the number of 8q copies was not associated with survival when comparing early (P = 0.97) versus late (P = 0.23) metastases group. In contrast, the presence of 8q gain (86%) was correlated with a decreased survival in BAP1 MUT UM (P = 0.013). Conclusions: We did not find a correlation between 8q gain and early-onset metastasis in SF3B1 MUT tumors. Gain of 8q has no additional predic

Purpose: Uveal melanoma (UM) has a high propensity to metastasize. Prognosis is associated with specific driver mutations and copy number variations (CNVs), but limited primary tumor tissue is available for molecular characterization due to eye-sparing irradiation treatment. This study aimed to assess the rise in circulating tumor DNA (ctDNA) levels in UM and evaluate its efficacy for CNV-profiling of patients with UM. Methods:In a pilot study, we assessed ctDNA levels in the blood of patients with UM (n = 18) at various time points, including the time of diagnosis (n = 13), during fractionated stereotactic radiotherapy (fSRT) treatment (n = 6), and upon detection of metastatic disease (n = 13). Shallow whole-genome sequencing (sWGS) combined with in silico size-selection was used to identify prognostically relevant CNVs in patients with UM (n = 26) from peripheral blood retrieved at the time of diagnosis (n = 9), during fSRT (n = 5), during post-treatment follow-up (n = 4), metastasis detection (n = 6), and metastasis follow-up (n = 4). Results: A total of 34 patients had blood analyzed for ctDNA detection (n = 18) and/or CNV analysis (n = 26) at various time points. At the time of diagnosis, 5 of 13 patients (38%) had detectable ctDNA (median = 0 copies/mL). Upon detection of metastatic disease, ctDNA was detected in 10 of 13 patients (77%) and showed increased ctDNA levels (median = 24 copies/mL, P < 0.01). Among the six patients analyzed during fSRT, three (50%) patients had detectable ctDNA at baseline and three of six (50%) patients had undetectable levels of ctDNA. During the fSRT regimen, ctDNA levels remained unchanged (P > 0.05). The ctDNA fractions were undetectable to low in localized disease, and sWGS did not elucidate chromosome 3 status from blood samples. However, in 7 of 10 (70%) patients with metastases, the detection of chromosome 3 loss corresponded to the high metastatic-risk class

Purpose: The worldwide incidence of ocular melanoma (OM), uveal melanoma (UM), and conjunctival melanoma has last been reported on 15 years ago. Recently, light iris color and four specific single-nucleotide-polymorphisms (SNPs) have been identified as a UM-risk factor. Furthermore, six iris color predicting SNPs have been discovered (IrisPlex). Interestingly, two of these (rs129138329 and rs12203592) are also UM-risk factors. We collected worldwide incidence data of OM and investigated its correlations with iris color, IrisPlex SNPs, and UM-risk SNPs. Methods: Cases of OM, as defined by the International Classification of Diseases Oncology C69 (eye), 8720/3 to 8790/3 (malignant melanoma), and 8000 to 8005 (malignant neoplasm), between 1988 and 2012, were extracted from the Cancer Incidence in Five Continents. Incidence rates were age-standardized and their trends were analyzed with joinpoint regression and age period cohort modeling. Frequencies for each country of iris color, IrisPlex SNPs, and UM-risk SNPs were collected from the literature. Results:Incidence rates were generally ≥8.0 cases per million person-years in Northern Europe, Western Europe, and Oceania; 2.0 to 7.9 in North America, Eastern Europe, and Southern Europe; and <2.0 in South America, Asia, and Africa. OM incidence correlated with latitude (r = 0.77, P ≤ 0.001) and is expressed as a north-to-south decreasing gradient in Europe. SNP rs12913832 correlated with OM incidence (r = 0.83, P ≤ 0.001), blue iris color (r = 0.56, P ≤ 0.05), green iris color (r = 0.51, P ≤ 0.05), and brown iris color (r = -0.64, P ≤ 0.01). Trends were stable for most countries (28/35). Conclusions: OM incidence is highest in populations of European ancestry and lowest in populations of Asian and African ancestry. Overall, trends are stable, and the spatial correlation among OM incidence, iris color, and rs12913832 may support the role of p

Currently there is a global lack of consensus about the best treatment for asymptomatic congenital pulmonary airway malformation (CPAM) patients. The somatic KRAS mutations commonly found in adult lung cancer combined with mucinous proliferations are sometimes found in CPAM. For this risk of developing malignancy, 70% of paediatric surgeons perform a resection for asymptomatic CPAM. In order to stratify these patients into high-and low-risk groups for developing malignancy, a minimally invasive diagnostic method is needed, for example targeted molecular imaging. A prerequisite for this technique is a cell membrane bound target. The aim of this study was to review the literature to identify potential targets for molecular imaging in CPAM patients and perform a first step to validate these findings. A systematic search was conducted to identify possible targets in CPAM and adenocarcinoma in situ (AIS) patients. The most interesting targets were evaluated with immunofluorescent staining in adjacent lung tissue, KRAS+ CPAM tissue and KRAS– CPAM tissue. In 185 included studies, 143 possible targets were described, of which 20 targets were upregulated and membrane-bound. Six of them were also upregulated in lung AIS tissue (CEACAM5, E-cadherin, EGFR, ERBB2, ITGA2 and MUC1) and as such of possible interest. Validating studies showed that MUC1 is a potential interesting target. This study provides an extensive overview of all known potential targets in CPAM that might identify those patients at risk for malignancy and conducted the first step towards validation, identifying MUC1 as the most promising target.

Uveal melanomas (UM) are detected earlier. Consequently, tumors are smaller, allowing for novel eye-preserving treatments. This reduces tumor tissue available for genomic profiling. Additionally, these small tumors can be hard to differentiate from nevi, creating the need for minimally invasive detection and prognostication. Metabolites show promise as minimally invasive detection by resembling the biological phenotype. In this pilot study, we determined metabolite patterns in the peripheral blood of UM patients (n = 113) and controls (n = 46) using untargeted metabolomics. Using a random forest classifier (RFC) and leave-one-out cross-validation, we confirmed discriminatory metabolite patterns in UM patients compared to controls with an area under the curve of the receiver operating characteristic of 0.99 in both positive and negative ion modes. The RFC and leave-one-out cross-validation did not reveal discriminatory metabolite patterns in high-risk versus low-risk of metastasizing in UM patients. Ten-time repeated analyses of the RFC and LOOCV using 50% randomly distributed samples showed similar results for UM patients versus controls and prognostic groups. Pathway analysis using annotated metabolites indicated dysregulation of several processes associated with malignancies. Consequently, minimally invasive metabolomics could potentially allow for screening as it distinguishes metabolite patterns that are putatively associated with oncogenic processes in the peripheral blood plasma of UM patients from controls at the time of diagnosis.

INTRODUCTION: Consensus is lacking on the optimal management of asymptomatic congenital pulmonary airway malformation (CPAM). For future studies, the CONNECT consortium (the COllaborative Neonatal Network for the first European CPAM Trial)-an international collaboration of specialised caregivers-has established consensus on a core outcome set of outcome parameters concerning respiratory insufficiency, surgical complications, mass effect and multifocal disease. These outcome parameters have been incorporated in the CONNECT trial, a randomised controlled trial which, in order to develop evidence-based practice, aims to compare conservative and surgical management of patients with an asymptomatic CPAM.METHODS AND ANALYSIS: Children are eligible for inclusion after the CPAM diagnosis has been confirmed on postnatal chest CT scan and they remain asymptomatic. On inclusion, children are randomised to receive either conservative or surgical management. Subsequently, children in both groups are enrolled into a standardised, 5-year follow-up programme with three visits, including a repeat chest CT scan at 2.5 years and a standardised exercise tolerance test at 5 years.The primary outcome is exercise tolerance at age 5 years, measured according to the Bruce treadmill protocol. Secondary outcome measures are molecular genetic diagnostics, validated questionnaires-on parental anxiety, quality of life and healthcare consumption-, repeated imaging and pulmonary morbidity during follow-up, as well as surgical complications and histopathology. This trial aims to end the continuous debate surrounding the optimal management of asymptomatic CPAM. ETHICS AND DISSEMINATION: This study is being conducted in accordance with the Declaration of Helsinki. The Medical Ethics Review Board of Erasmus University Medical Centre Rotterdam, The Netherlands, has approved this protocol (MEC-2022-0441). Results will be disseminated through pee

Uveal melanoma (UM) is the second most frequent type of melanoma. Therapeutic options for UM favor minimally invasive techniques such as irradiation for vision preservation. As a consequence, no tumor material is obtained. Without available tissue, molecular analyses for gene expression, mutation or copy number analysis cannot be performed. Thus, proper patient stratification is impossible and patients' uncertainty about their prognosis rises. Minimally invasive techniques have been studied for prognostication in UM. Blood-based biomarker analysis has become more common in recent years; however, no clinically standardized protocol exists. This review summarizes insights in biomarker analysis, addressing new insights in circulating tumor cells, circulating tumor DNA, extracellular vesicles, proteomics, and metabolomics. Additionally, medical imaging can play a significant role in staging, surveillance, and prognostication of UM and is addressed in this review. We propose that combining multiple minimally invasive modalities using tumor biomarkers should be the way forward and warrant more attention in the coming years.

Uveal melanoma (UM) is the second most frequent type of melanoma. Therapeutic options for UM favor minimally invasive techniques such as irradiation for vision preservation. As a consequence, no tumor material is obtained. Without available tissue, molecular analyses for gene expression, mutation or copy number analysis cannot be performed. Thus, proper patient stratification is impossible and patients' uncertainty about their prognosis rises. Minimally invasive techniques have been studied for prognostication in UM. Blood-based biomarker analysis has become more common in recent years; however, no clinically standardized protocol exists. This review summarizes insights in biomarker analysis, addressing new insights in circulating tumor cells, circulating tumor DNA, extracellular vesicles, proteomics, and metabolomics. Additionally, medical imaging can play a significant role in staging, surveillance, and prognostication of UM and is addressed in this review. We propose that combining multiple minimally invasive modalities using tumor biomarkers should be the way forward and warrant more attention in the coming years.