RESUMEN
Reactivation of fetal hemoglobin expression alleviates the symptoms associated with ß-globinopathies, severe hereditary diseases with significant global health implications due to their high morbidity and mortality rates. The symptoms emerge following the postnatal transition from fetal-to-adult hemoglobin expression. Extensive research has focused on inducing the expression of the fetal γ-globin subunit to reverse this switch and ameliorate these symptoms. Despite decades of research, only one compound, hydroxyurea, found its way to the clinic as an inducer of fetal hemoglobin. Unfortunately, its efficacy varies among patients, highlighting the need for more effective treatments. Erythroid cell lines have been instrumental in the pursuit of both pharmacological and genetic ways to reverse the postnatal hemoglobin switch. Here, we describe the first endogenously tagged fetal hemoglobin reporter cell line based on the adult erythroid progenitor cell line HUDEP2. Utilizing CRISPR-Cas9-mediated knock-in, a bioluminescent tag was integrated at the HBG1 gene. Subsequent extensive characterization confirmed that the resulting reporter cell line closely mirrors the HUDEP2 characteristics and that the cells report fetal hemoglobin induction with high sensitivity and specificity. This novel reporter cell line is therefore highly suitable for evaluating genetic and pharmacologic strategies to induce fetal hemoglobin. Furthermore, it provides an assay compatible with high-throughput drug screening, exemplified by the identification of a cluster of known fetal hemoglobin inducers in a pilot study. This new tool is made available to the research community, with the aspiration that it will accelerate the search for safer and more effective strategies to reverse the hemoglobin switch.
RESUMEN
In patients with proximal hypospadias, often no genetic cause is identified despite extensive genetic testing. Many genes involved in sex development encode transcription factors with strict timing and dosing of the gene products. We hypothesised that there might be recurrent differences in DNA methylation in boys with hypospadias and that these might differ between patients born small versus appropriate for gestational age. Genome-wide Methylated DNA sequencing (MeD-seq) was performed on 32bp LpnPI restriction enzyme fragments after RE-digestion in leucocytes from 16 XY boys with unexplained proximal hypospadias, one with an unexplained XX testicular disorder/difference of sex development (DSD) and twelve, healthy, sex- and age-matched controls. Five of seven differentially methylated regions (DMRs) between patients and XY controls were in the Long Intergenic Non-Protein Coding RNA 665 (LINC00665; CpG24525). Three patients showed hypermethylation of MAP3K1. Finally, no DMRs in XX testicular DSD associated genes were identified in the XX boy versus XX controls. In conclusion, we observed no recognizable epigenetic signature in 16 boys with XY proximal hypospadias and no difference between children born small versus appropriate for gestational age. Comparison to previous methylation studies in individuals with hypospadias did not show consistent findings, possibly due to the use of different inclusion criteria, tissues and methods.
Asunto(s)
Metilación de ADN , Hipospadias , Humanos , Masculino , Hipospadias/genética , Proyectos Piloto , Epigénesis Genética , Islas de CpG , ARN Largo no Codificante/genética , Niño , Preescolar , Lactante , Estudio de Asociación del Genoma Completo , Estudios de Casos y ControlesRESUMEN
AIMS: Uveal melanoma has a high propensity to metastasize. Prognosis is associated with specific driver mutations and copy number variations, and these can only be obtained after genetic testing. In this study we evaluated the efficacy of patient outcome prediction using deep learning on haematoxylin and eosin (HE)-stained primary uveal melanoma slides in comparison to molecular testing. METHODS: In this retrospective study of patients with uveal melanoma, 113 patients from the Erasmus Medical Centre who underwent enucleation had tumour tissue analysed for molecular classification between 1993 and 2020. Routine HE-stained slides were scanned to obtain whole-slide images (WSI). After annotation of regions of interest, tiles of 1024 × 1024 pixels were extracted at a magnification of 40×. An ablation study to select the best-performing deep-learning model was carried out using three state-of-the-art deep-learning models (EfficientNet, Vision Transformer, and Swin Transformer). RESULTS: Deep-learning models were subjected to a training cohort (n = 40), followed by a validation cohort (n = 20), and finally underwent a test cohort (n = 48). A k-fold cross-validation (k = 3) of validation and test cohorts (n = 113 of three classes: BAP1, SF3B1, EIF1AX) demonstrated Swin Transformer as the best-performing deep-learning model to predict molecular subclasses based on HE stains. The model achieved an accuracy of 0.83 ± 0.09 on the validation cohort and 0.75 ± 0.04 on the test cohort. Within the subclasses, this model correctly predicted 70% BAP1-mutated, 61% SF3B1-mutated and 80% EIF1AX-mutated UM in the test set. CONCLUSIONS: This study showcases the potential of the deep-learning methodology for predicting molecular subclasses in a multiclass manner using HE-stained WSI. This development holds promise for advanced prognostication of UM patients without the need of molecular or immunohistochemical testing. Additionally, this study suggests there are distinct histopathological features per subclass; mainly utilizing epithelioid cellular morphology for BAP1-classification, but an unknown feature distinguishes EIF1AX and SF3B1.
RESUMEN
Purpose: To explore the genetic background of choroidal and ciliary body melanoma among children and young adults, with special focus on BAP1 germline variants in this age group. Methods: Patients under the age of 25 and with confirmed choroidal or ciliary body melanoma were included in this retrospective, multicenter observational study. Nuclear BAP1 immunopositivity was used to evaluate the presence of functional BAP1 in the tumor. Next-generation sequencing using Ion Torrent platform was used to determine pathogenic variants of BAP1, EIF1AX, SF3B1, GNAQ and GNA11 and chromosome 3 status in the tumor or in DNA extracted from blood or saliva. Survival was analyzed using Kaplan-Meier estimates. Results: The mean age at diagnosis was 17 years (range 5.0-24.8). A germline BAP1 pathogenic variant was identified in an 18-year-old patient, and a somatic variant, based mainly on immunohistochemistry, in 13 (42%) of 31 available specimens. One tumor had a somatic SF3B1 pathogenic variant. Disomy 3 and the absence of a BAP1 pathogenic variant in the tumor predicted the longest metastasis-free survival. Males showed longer metastasis-free survival than females (P = 0.018). Conclusions: We did not find a stronger-than-average BAP1 germline predisposition for choroidal and ciliary body melanoma among children and young adults compared to adults. Males had a more favorable survival and disomy 3, and the absence of a BAP1 mutation in the tumor tissue predicted the most favorable metastasis-free survival. A BAP1 germline pathogenic variant was identified in one patient (1%), and a somatic variant based mainly on immunohistochemistry in 13 (42%).
Asunto(s)
Melanoma , Neoplasias de la Úvea , Adolescente , Niño , Preescolar , Femenino , Humanos , Masculino , Adulto Joven , Cuerpo Ciliar , Melanoma/genética , Estudios Retrospectivos , Neoplasias de la Úvea/genéticaRESUMEN
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. Conclusions: The rise in ctDNA levels observed in patients with UM harboring metastases suggests its potential utility for CNV profiling. These findings highlight the potential of using ctDNA for metastasis detection and patient inclusion in therapeutic studies targeting metastatic UM.
Asunto(s)
ADN Tumoral Circulante , Melanoma , Neoplasias de la Úvea , Humanos , ADN Tumoral Circulante/genética , Variaciones en el Número de Copia de ADN , Proyectos Piloto , BiomarcadoresRESUMEN
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 (SF3B1MUT) tumors display structural chromosomal anomalies and frequently show a partial gain of chromosome 8qter. A recent subset of SF3B1MUT UM with early-onset metastases has been identified, prompting the investigation of the relationship between survival, 8q gain, and SF3B1MUT 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 SF3B1MUT tumors and 211 patients with BRCA1 associated protein 1 (BAP1)-mutated (BAP1MUT) 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 SF3B1MUT UM (81%) had chromosome 8q gain (3 copies, 78%; 4 copies, 22%). Kaplan-Meier analysis of SF3B1MUT 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 BAP1MUT UM (P = 0.013). Conclusions: We did not find a correlation between 8q gain and early-onset metastasis in SF3B1MUT tumors. Gain of 8q has no additional predictive value in SF3B1MUT tumors. In contrast, 8q gain is predictive of a worse prognosis in patients with BAP1MUT tumors. Thus, gain of chromosome 8q has additional predictive value for BAP1MUT tumors, but not for SF3B1MUT tumors. Financial Disclosures: The author(s) have no proprietary or commercial interest in any materials discussed in this article.
RESUMEN
Despite extensive research and refined therapeutic options, the survival for metastasized uveal melanoma (UM) patients has not improved significantly. UM, a malignant tumor originating from melanocytes in the uveal tract, can be asymptomatic and small tumors may be detected only during routine ophthalmic exams; making early detection and treatment difficult. UM is the result of a number of characteristic somatic alterations which are associated with prognosis. Although UM morphology and biology have been extensively studied, there are significant gaps in our understanding of the early stages of UM tumor evolution and effective treatment to prevent metastatic disease remain elusive. A better understanding of the mechanisms that enable UM cells to thrive and successfully metastasize is crucial to improve treatment efficacy and survival rates. For more than forty years, animal models have been used to investigate the biology of UM. This has led to a number of essential mechanisms and pathways involved in UM aetiology. These models have also been used to evaluate the effectiveness of various drugs and treatment protocols. Here, we provide an overview of the molecular mechanisms and pharmacological studies using mouse and zebrafish UM models. Finally, we highlight promising therapeutics and discuss future considerations using UM models such as optimal inoculation sites, use of BAP1mut-cell lines and the rise of zebrafish models.
Asunto(s)
Melanoma , Neoplasias de la Úvea , Humanos , Animales , Ratones , Pez Cebra , Melanoma/tratamiento farmacológico , Neoplasias de la Úvea/genética , Neoplasias de la Úvea/metabolismoRESUMEN
Craniosynostosis may present in isolation, 'non-syndromic', or with additional congenital anomalies/neurodevelopmental disorders, 'syndromic'. Clinical focus shifted from confirming classical syndromic cases to offering genetic testing to all craniosynostosis patients. This retrospective study assesses diagnostic yield of molecular testing by investigating prevalences of chromosomal and monogenic (likely) pathogenic variants in an 11-year cohort of 1020 craniosynostosis patients. 502 children underwent genetic testing. Pathogenic variants were identified in 174 patients (35%). Diagnostic yield was significantly higher in syndromic craniosynostosis (62%) than in non-syndromic craniosynostosis (6%). Before whole exome sequencing (WES) emerged, single-gene testing was performed using Sanger sequencing or multiplex ligation-dependent probe amplification (MLPA). Diagnostic yield was 11% and was highest for EFNB1, FGFR2, FGFR3, and IL11RA. Diagnostic yield for copy number variant analysis using microarray was 8%. From 2015 onwards, the WES craniosynostosis panel was implemented, with a yield of 10%. In unsolved, mainly syndromic, cases suspected of a genetic cause, additional WES panels (multiple congenital anomalies (MCA)/intellectual disability (ID)) or open exome analysis were performed with an 18% diagnostic yield. To conclude, microarray and the WES craniosynostosis panel are key to identifying pathogenic variants. in craniosynostosis patients. Given the advances in genetic diagnostics, we should look beyond the scope of the WES craniosynostosis panel and consider extensive genetic diagnostics (e.g. open exome sequencing, whole genome sequencing, RNA sequencing and episignature analysis) if no diagnosis is obtained through microarray and/or WES craniosynostosis panel. If parents are uncomfortable with more extensive diagnostics, MCA or ID panels may be considered.
RESUMEN
BACKGROUND: Approximately 50% of uveal melanoma (UM) patients will develop metastatic disease depending on the genetic features of the primary tumour. Patients need 3-12 monthly scans, depending on their prognosis, which is costly and often non-specific. Circulating tumour DNA (ctDNA) quantification could serve as a test to detect and monitor patients for early signs of metastasis and therapeutic response. METHODS: We assessed ctDNA as a biomarker in three distinct UM cohorts using droplet-digital PCR: (A) a retrospective analysis of primary UM patients to predict metastases; (B) a prospective analysis of UM patients after resolution of their primary tumour for early detection of metastases; and (C) monitoring treatment response in metastatic UM patients. RESULTS: Cohort A: ctDNA levels were not associated with the development of metastases. Cohort B: ctDNA was detected in 17/25 (68%) with radiological diagnosis of metastases. ctDNA was the strongest predictor of overall survival in a multivariate analysis (HR = 15.8, 95% CI 1.7-151.2, p = 0.017). Cohort C: ctDNA monitoring of patients undergoing immunotherapy revealed a reduction in the levels of ctDNA in patients with combination immunotherapy. CONCLUSIONS: Our proof-of-concept study shows the biomarker feasibility potential of ctDNA monitoring in for the clinical management of uveal melanoma patients.
Asunto(s)
ADN Tumoral Circulante , Melanoma , Humanos , ADN Tumoral Circulante/genética , Estudios Retrospectivos , Melanoma/patología , Biomarcadores , Biomarcadores de Tumor/genéticaRESUMEN
Alveolar capillary dysplasia with misalignment of pulmonary veins (ACDMPV) is a lethal congenital lung disorder that presents shortly after birth with respiratory failure and therapy-resistant pulmonary hypertension. It is associated with heterozygous point mutations and genomic deletions that involve the FOXF1 gene or its upstream regulatory region. Patients are unresponsive to the intensive treatment regimens and suffer unnecessarily because ACDMPV is not always timely recognized and histologic diagnosis is invasive and time consuming. Here, we demonstrate the usefulness of a noninvasive, fast genetic test for FOXF1 variants that we previously developed to rapidly diagnose ACDMPV and reduce the time of hospitalization.
Asunto(s)
Síndrome de Circulación Fetal Persistente , Alveolos Pulmonares/anomalías , Recién Nacido , Humanos , Síndrome de Circulación Fetal Persistente/diagnóstico , Síndrome de Circulación Fetal Persistente/genética , Síndrome de Circulación Fetal Persistente/patología , Relevancia Clínica , Alveolos Pulmonares/patología , Factores de Transcripción Forkhead/genéticaRESUMEN
Purpose: Myopia (nearsightedness) is a condition in which a refractive error (RE) affects vision. Although common variants explain part of the genetic predisposition (18%), most of the estimated 70% heritability is missing. Here, we investigate the contribution of rare genetic variation because this might explain more of the missing heritability in the more severe forms of myopia. In particular, high myopia can lead to blindness and has a tremendous impact on a patient and at the societal level. The exact molecular mechanisms behind this condition are not yet completely unraveled, but whole genome sequencing (WGS) studies have the potential to identify novel (rare) disease genes, explaining the high heritability. Design: Cross-sectional study performed in the Netherlands. Participants: We investigated 159 European patients with high myopia (RE > -10 diopters). Methods: We performed WGS using a stepwise filtering approach and burden analysis. The contribution of common variants was calculated as a genetic risk score (GRS). Main Outcome Measures: Rare variant burden, GRS. Results: In 25% (n = 40) of these patients, there was a high (> 75th percentile) contribution of common predisposing variants; that is, these participants had higher GRSs. In 7 of the remaining 119 patients (6%), deleterious variants in genes associated with known (ocular) disorders, such as retinal dystrophy disease (prominin 1 [PROM1]) or ocular development (ATP binding cassette subfamily B member 6 [ABCB6], TGFB induced factor homeobox 1 [TGIF1]), were identified. Furthermore, without using a gene panel, we identified a high burden of rare variants in 8 novel genes associated with myopia. The genes heparan sulfate 6-O-sulfotransferase 1 (HS6ST1) (proportion in study population vs. the Genome Aggregation Database (GnomAD) 0.14 vs. 0.03, P = 4.22E-17), RNA binding motif protein 20 (RBM20) (0.15 vs. 0.06, P = 4.98E-05), and MAP7 domain containing 1 (MAP7D1) (0.19 vs. 0.06, P = 1.16E-10) were involved in the Wnt signaling cascade, melatonin degradation, and ocular development and showed most biologically plausible associations. Conclusions: We found different contributions of common and rare variants in low and high grade myopia. Using WGS, we identified some interesting candidate genes that could explain the high myopia phenotype in some patients. Financial Disclosures: The author(s) have no proprietary or commercial interest in any materials discussed in this article.
RESUMEN
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.
Asunto(s)
Melanoma , Neoplasias de la Úvea , Humanos , Proyectos Piloto , Melanoma/genética , Neoplasias de la Úvea/diagnóstico , Neoplasias de la Úvea/genética , FenotipoRESUMEN
Purpose: To determine whether peripheral blood leukocyte numbers and serum markers of inflammation can be used to predict which patients with primary uveal melanoma will develop metastasis. Design: Retrospective study. Participants: Medical records of patients with uveal melanoma (UM) who received treatment for primary UM between February 1992 and December 2020 at the Erasmus University Medical Center (Rotterdam, The Netherlands) and the Rotterdam Eye Hospital (Rotterdam, The Netherlands) were reviewed. Methods: Inclusion criteria were the presence of a melanoma of the choroid or ciliary body and the availability of data from peripheral blood samples taken before treatment of the melanoma. Data including patient demographics, C-reactive protein (CRP) levels; erythrocyte sedimentation rate (ESR); number of leukocytes, neutrophils, monocytes, and lymphocytes; and histopathologic findings were obtained from medical records. Neutrophil-to-lymphocyte ratio (NLR) and lymphocyte-to-monocyte ratio (LMR) were calculated. Main Outcome Measures: Metastasis-free survival. Results: Of the 807 patients with UM, serum and leukocyte data were available for 183 of them at the time of primary tumor treatment. In the total group, no correlation was found between ESR before treatment; the number of leukocytes; percentages of neutrophils, monocytes, and lymphocytes; or NLR or LMR values and any of the clinical characteristics or metastasis-free survival. Among patients who underwent enucleation, those with negative BAP1 findings showed significantly lower numbers of leukocytes (P < 0.05). In the entire cohort, a significant association was found between high CRP levels and longer metastasis-free survival (MFS; P = 0.049). Conclusions: The total blood leukocyte number was related to loss of BAP1 staining in patients who underwent enucleation, with lower leukocyte counts correlating with absent BAP1 staining. Higher CRP levels were associated with a longer MFS in the entire cohort. Neither the NLR nor the LMR is a good predictor for metastasis developing in patients with UM.
RESUMEN
Uveal melanoma (UM) is a deadly ocular malignancy, originating from uveal melanocytes. Although much is known regarding prognostication in UM, the exact mechanism of metastasis is mostly unknown. Metastatic tumor cells are known to express a more stem-like RNA profile which is seen often in cell-specific embryonic development to induce tumor progression. Here, we identified novel transcription regulators by reanalyzing publicly available single cell RNA sequencing experiments. We identified five transcription regulators of interest: ELL2, KDM5B, REXO4, RBFOX2 and FOXD1. Our most significant finding is FOXD1, as this gene is nearly exclusively expressed in high-risk UM and its expression is associated with a poor prognosis. Even within the BAP1-mutated UM, the expression of FOXD1 is correlated with poor survival. FOXD1 is a novel factor which could potentially be involved in the metastatic capacity of high-risk UM. Elucidating the function of FOXD1 in UM could provide insight into the malignant transformation of uveal melanocytes, especially in high-risk UM.
RESUMEN
Herein, we report on a large Polish family presenting with a classical triphalangeal thumb-polysyndactyly syndrome (TPT-PS). This rare congenital limb anomaly is generally caused by microduplications encompassing the Sonic Hedgehog (SHH) limb enhancer, termed the zone of polarizing activity (ZPA) regulatory sequence (ZRS). Recently, a pathogenic variant in the pre-ZRS (pZRS), a conserved sequence located near the ZRS, has been described in a TPT-PS Dutch family. We performed targeted ZRS sequencing, array comparative genomic hybridization, and whole-exome sequencing. Next, we sequenced the recently described pZRS region. Finally, we performed a circular chromatin conformation capture-sequencing (4C-seq) assay on skin fibroblasts of one affected family member and control samples to examine potential alterations in the SHH regulatory domain and functionally characterize the identified variant. We found that all affected individuals shared a recently identified pathogenic point mutation in the pZRS region: NC_000007.14:g.156792782C>G (GRCh38/hg38), which is the same as in the Dutch family. The results of 4C-seq experiments revealed increased interactions within the whole SHH regulatory domain (SHH-LMBR1 TAD) in the patient compared to controls. Our study expands the number of TPT-PS families carrying a pathogenic alteration of the pZRS and underlines the importance of routine pZRS sequencing in the genetic diagnostics of patients with TPT-PS or similar phenotypes. The pathogenic mutation causative for TPT-PS in our patient gave rise to increased interactions within the SHH regulatory domain in yet unknown mechanism.
Asunto(s)
Anomalías Congénitas , Proteínas Hedgehog , Disostosis Mandibulofacial , Polidactilia , Hibridación Genómica Comparativa , Anomalías Congénitas/genética , Elementos de Facilitación Genéticos , Proteínas Hedgehog/genética , Humanos , Disostosis Mandibulofacial/genética , Mutación , Linaje , PulgarRESUMEN
Induced pluripotent stem cell (iPSC)-derived kidney organoids are a potential tool for the regeneration of kidney tissue. They represent an early stage of nephrogenesis and have been shown to successfsully vascularize and mature further in vivo. However, there are concerns regarding the long-term safety and stability of iPSC derivatives. Specifically, the potential for tumorigenesis may impede the road to clinical application. To study safety and stability of kidney organoids, we analyzed their potential for malignant transformation in a teratoma assay and following long-term subcutaneous implantation in an immune-deficient mouse model. We did not detect fully functional residual iPSCs in the kidney organoids as analyzed by gene expression analysis, single-cell sequencing and immunohistochemistry. Accordingly, kidney organoids failed to form teratoma. Upon long-term subcutaneous implantation of whole organoids in immunodeficient IL2Ry-/-RAG2-/- mice, we observed tumor formation in 5 out of 103 implanted kidney organoids. These tumors were composed of WT1+CD56+ immature blastemal cells and showed histological resemblance with Wilms tumor. No genetic changes were identified that contributed to the occurrence of tumorigenic cells within the kidney organoids. However, assessment of epigenetic changes revealed a unique cluster of differentially methylated genes that were also present in undifferentiated iPSCs. We discovered that kidney organoids have the capacity to form tumors upon long-term implantation. The presence of epigenetic modifications combined with the lack of environmental cues may have caused an arrest in terminal differentiation. Our results indicate that the safe implementation of kidney organoids should exclude the presence of pro-tumorigenic methylation in kidney organoids.
Asunto(s)
Células Madre Pluripotentes Inducidas , Teratoma , Animales , Diferenciación Celular , Células Madre Pluripotentes Inducidas/metabolismo , Riñón/patología , Ratones , Organogénesis , Organoides/metabolismo , Teratoma/patologíaRESUMEN
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.
RESUMEN
The prevalence of Barrett's esophagus (BE) in adults born with esophageal atresia (EA) is four times higher than in the general population and presents at a younger age (34 vs. 60 years). This is (partly) a consequence of chronic gastroesophageal reflux. Given the overlap between genes and pathways involved in foregut and BE development, we hypothesized that EA patients have an intrinsic predisposition to develop BE. Transcriptomes of Esophageal biopsies of EA patients with BE (n = 19, EA/BE); EA patients without BE (n = 44, EA-only) and BE patients without EA (n = 10, BE-only) were compared by RNA expression profiling. Subsequently, we simulated a reflux episode by exposing fibroblasts of 3 EA patients and 3 controls to acidic conditions. Transcriptome responses were compared to the differential expressed transcripts in the biopsies. Predisposing single nucleotide polymorphisms, associated with BE, were slightly increased in EA/BE versus BE-only patients. RNA expression profiling and pathway enrichment analysis revealed differences in retinoic acid metabolism and downstream signaling pathways and inflammatory, stress response and oncological processes. There was a similar effect on retinoic acid signaling and immune response in EA patients upon acid exposure. These results indicate that epithelial tissue homeostasis in EA patients is more prone to acidic disturbances.
RESUMEN
Approximately 25% of all uveal melanoma (UM) contain driver mutations in the gene encoding the spliceosome factor SF3B1, and whilst patients with such SF3B1 mutations generally have an intermediate risk on developing metastatic disease, a third of these patients develop early metastasis within 5 years after diagnosis. We therefore investigated whether clinical and/or genetic variables could be indicative of short progression-free survival (PFS < 60 months) or long PFS (PFS ≥ 60 months) for SF3B1-mutated (SF3B1mut) UM patients. We collected 146 SF3B1mut UM from our Rotterdam Ocular Melanoma Studygroup (ROMS) database and external published datasets. After stratification of all SF3B1mut UM using short PFS vs. long PFS, only largest tumor diameter (LTD) was significantly larger (mean: 17.7 mm (±2.8 SD) in the short PFS SF3B1mut group vs. the long PFS group (mean: 14.7 (±3.7 SD, p = 0.001). Combined ROMS and The Cancer Genome Atlas (TCGA) transcriptomic data were evaluated, and we identified SF3B1mut-specific canonical transcripts (e.g., a low expression of ABHD6 indicative for early-onset metastatic disease) or distinct expression of SF3B1mut UM aberrant transcripts, indicative of early- or late-onset or no metastatic SF3B1mut UM.
RESUMEN
Uveal melanoma (UM) is an aggressive intra-ocular cancer with a strong tendency to metastasize. Metastatic UM is associated with mutations in BAP1 and SF3B1, however only little is known about the epigenetic modifications that arise in metastatic UM. In this study we aim to unravel epigenetic changes contributing to UM metastasis using a new genome-wide methylation analysis technique that covers over 50% of all CpG's. We identified aberrant methylation contributing to BAP1 and SF3B1-mediated UM metastasis. The methylation data was integrated with expression data and surveyed in matched UM metastases from the liver, skin and bone. UM metastases showed no commonly shared novel epigenetic modifications, implying that epigenetic changes contributing to metastatic spreading and colonization in distant tissues occur early in the development of UM and epigenetic changes that occur after metastasis are mainly patient-specific. Our findings reveal a plethora of epigenetic modifications in metastatic UM and its metastases, which could subsequently result in aberrant repression or activation of many tumor-related genes. This observation points towards additional layers of complexity at the level of gene expression regulation, which may explain the low mutational burden of UM.