RESUMEN
PRAME is a CUL2 ubiquitin ligase subunit that is normally expressed in the testis but becomes aberrantly overexpressed in many cancer types in association with aneuploidy and metastasis. Here, we show that PRAME is expressed predominantly in spermatogonia around the time of meiotic crossing-over in coordination with genes mediating DNA double strand break repair. Expression of PRAME in somatic cells upregulates pathways involved in meiosis, chromosome segregation and DNA repair, and it leads to increased DNA double strand breaks, telomere dysfunction and aneuploidy in neoplastic and non-neoplastic cells. This effect is mediated at least in part by ubiquitination of SMC1A and altered cohesin function. PRAME expression renders cells susceptible to inhibition of PARP1/2, suggesting increased dependence on alternative base excision repair pathways. These findings reveal a distinct oncogenic function of PRAME that can be targeted therapeutically in cancer.
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Melanoma , Neoplasias de la Úvea , Masculino , Humanos , Melanoma/genética , Reparación del ADN/genética , ADN , Inestabilidad Genómica , Aneuploidia , Meiosis , Antígenos de Neoplasias/metabolismoRESUMEN
PURPOSE: Validated and accurate prognostic testing is critical for precision medicine in uveal melanoma (UM). Our aims were to (1) prospectively validate an integrated prognostic classifier combining a 15-gene expression profile (15-GEP) and PRAME RNA expression and (2) identify clinical variables that enhance the prognostic accuracy of the 15-GEP/PRAME classifier. MATERIALS AND METHODS: This study included 1,577 patients with UM of the choroid and/or ciliary body who were enrolled in the Collaborative Ocular Oncology Group Study Number 2 (COOG2) and prospectively monitored across 26 North American centers. Test results for 15-GEP (class 1 or class 2) and PRAME expression status (negative or positive) were available for all patients. The primary end point was metastasis-free survival (MFS). RESULTS: 15-GEP was class 1 in 1,082 (68.6%) and class 2 in 495 (31.4%) patients. PRAME status was negative in 1,106 (70.1%) and positive in 471 (29.9%) patients. Five-year MFS was 95.6% (95% CI, 93.9 to 97.4) for class 1/PRAME(-), 80.6% (95% CI, 73.9 to 87.9) for class 1/PRAME(+), 58.3% (95% CI, 51.1 to 66.4) for class 2/PRAME(-), and 44.8% (95% CI, 37.9 to 52.8) for class 2/PRAME(+). By multivariable Cox proportional hazards analysis, 15-GEP was the most important independent predictor of MFS (hazard ratio [HR], 5.95 [95% CI, 4.43 to 7.99]; P < .001), followed by PRAME status (HR, 1.82 [95% CI, 1.42 to 2.33]; P < .001). The only clinical variable demonstrating additional prognostic value was tumor diameter. CONCLUSION: In the largest prospective multicenter prognostic biomarker study performed to date in UM to our knowledge, the COOG2 study validated the superior prognostic accuracy of the integrated 15-GEP/PRAME classifier over 15-GEP alone and clinical prognostic variables. Tumor diameter was found to be the only clinical variable to provide additional prognostic information. This prognostic classifier provides an advanced resource for risk-adjusted metastatic surveillance and adjuvant trial stratification in patients with UM.
RESUMEN
PRAME is a CUL2 ubiquitin ligase subunit that is normally expressed in the testis but becomes aberrantly overexpressed in many cancer types in association with aneuploidy and metastasis. Here, we show that PRAME is expressed predominantly in spermatogonia around the time of meiotic crossing-over in coordination with genes mediating DNA double strand break repair. Expression of PRAME in somatic cells upregulates pathways involved in meiosis, chromosome segregation and DNA repair, and it leads to increased DNA double strand breaks, telomere dysfunction and aneuploidy in neoplastic and non-neoplastic cells. This effect is mediated at least in part by ubiquitination of SMC1A and altered cohesin function. PRAME expression renders cells susceptible to inhibition of PARP1/2, suggesting increased dependence on alternative base excision repair pathways. These findings reveal a distinct oncogenic function of PRAME than can be targeted therapeutically in cancer.
RESUMEN
Retinoblastoma (Rb) is a deadly childhood eye cancer that is classically initiated by inactivation of the RB1 tumor suppressor. Clinical management continues to rely on nonspecific chemotherapeutic agents that are associated with treatment resistance and toxicity. Here, we analyzed 103 whole exomes, 20 whole transcriptomes, 5 single-cell transcriptomes, and 4 whole genomes from primary Rb tumors to identify previously unknown Rb dependencies. Several recurrent genomic aberrations implicate estrogen-related receptor gamma (ESRRG) in Rb pathogenesis. RB1 directly interacts with and inhibits ESRRG, and RB1 loss uncouples ESRRG from negative regulation. ESRRG regulates genes involved in retinogenesis and oxygen metabolism in Rb cells. ESRRG is preferentially expressed in hypoxic Rb cells in vivo. Depletion or inhibition of ESRRG causes marked Rb cell death, which is exacerbated in hypoxia. These findings reveal a previously unidentified dependency of Rb cells on ESRRG, and they implicate ESRRG as a potential therapeutic vulnerability in Rb.
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Uveal melanoma (UM) is the most common primary cancer of the eye and is associated with a high rate of metastatic death. UM can be stratified into two main classes based on metastatic risk, with class 1 UM having a low metastatic risk and class 2 UM having a high metastatic risk. Class 2 UM have a distinctive genomic, transcriptomic, histopathologic, and clinical phenotype characterized by biallelic inactivation of the BAP1 tumor-suppressor gene, an immune-suppressive microenvironment enriched for M2-polarized macrophages, and poor response to checkpoint-inhibitor immunotherapy. To identify potential mechanistic links between BAP1 loss and immune suppression in class 2 UM, we performed an integrated analysis of UM samples, as well as genetically engineered UM cell lines and uveal melanocytes (UMC). Using RNA sequencing (RNA-seq), we found that the most highly upregulated gene associated with BAP1 loss across these datasets was PROS1, which encodes a ligand that triggers phosphorylation and activation of the immunosuppressive macrophage receptor MERTK. The inverse association between BAP1 and PROS1 in class 2 UM was confirmed by single-cell RNA-seq, which also revealed that MERTK was upregulated in CD163+ macrophages in class 2 UM. Using ChIP-seq, BAP1 knockdown in UM cells resulted in an accumulation of H3K27ac at the PROS1 locus, suggesting epigenetic regulation of PROS1 by BAP1. Phosphorylation of MERTK in RAW 264.7 monocyte-macrophage cells was increased upon coculture with BAP1-/- UMCs, and this phosphorylation was blocked by depletion of PROS1 in the UMCs. These findings were corroborated by multicolor immunohistochemistry, where class 2/BAP1-mutant UMs demonstrated increased PROS1 expression in tumor cells and increased MERTK phosphorylation in CD163+ macrophages compared with class 1/BAP1-wildtype UMs. Taken together, these findings provide a mechanistic link between BAP1 loss and the suppression of the tumor immune microenvironment in class 2 UMs, and they implicate the PROS1-MERTK pathway as a potential target for immunotherapy in UM.
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Albinism is a genetic defect characterized by a loss of pigmentation. The neurosensory retina, which is not pigmented, exhibits pathologic changes secondary to the loss of pigmentation in the retina pigment epithelium (RPE). How the loss of pigmentation in the RPE causes developmental defects in the adjacent neurosensory retina has not been determined, but offers a unique opportunity to investigate the interactions between these two important tissues. One of the genes that causes albinism encodes for an orphan GPCR (OA1) expressed only in pigmented cells, including the RPE. We investigated the function and signaling of OA1 in RPE and transfected cell lines. Our results indicate that OA1 is a selective L-DOPA receptor, with no measurable second messenger activity from two closely related compounds, tyrosine and dopamine. Radiolabeled ligand binding confirmed that OA1 exhibited a single, saturable binding site for L-DOPA. Dopamine competed with L-DOPA for the single OA1 binding site, suggesting it could function as an OA1 antagonist. OA1 response to L-DOPA was defined by several common measures of G-protein coupled receptor (GPCR) activation, including influx of intracellular calcium and recruitment of beta-arrestin. Further, inhibition of tyrosinase, the enzyme that makes L-DOPA, resulted in decreased PEDF secretion by RPE. Further, stimulation of OA1 in RPE with L-DOPA resulted in increased PEDF secretion. Taken together, our results illustrate an autocrine loop between OA1 and tyrosinase linked through L-DOPA, and this loop includes the secretion of at least one very potent retinal neurotrophic factor. OA1 is a selective L-DOPA receptor whose downstream effects govern spatial patterning of the developing retina. Our results suggest that the retinal consequences of albinism caused by changes in melanin synthetic machinery may be treated by L-DOPA supplementation.
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Albinismo/metabolismo , Dopaminérgicos/metabolismo , Proteínas del Ojo/metabolismo , Levodopa/metabolismo , Glicoproteínas de Membrana/metabolismo , Receptores Dopaminérgicos/metabolismo , Transducción de Señal/fisiología , Albinismo/genética , Animales , Comunicación Autocrina , Calcio/metabolismo , Línea Celular , Dopamina/metabolismo , Proteínas del Ojo/agonistas , Proteínas del Ojo/genética , Humanos , Ligandos , Glicoproteínas de Membrana/agonistas , Glicoproteínas de Membrana/genética , Monofenol Monooxigenasa/metabolismo , Factores de Crecimiento Nervioso/metabolismo , Epitelio Pigmentado Ocular/citología , Epitelio Pigmentado Ocular/metabolismo , Inhibidores de Proteasas/metabolismo , Receptores Dopaminérgicos/genética , Serpinas/metabolismo , Tirosina/metabolismoRESUMEN
Single-cell RNA sequencing (scRNA-seq) has been a transformative technology in many research fields. Dimensional reduction techniques such as UMAP and tSNE are used to visualize scRNA-seq data in two or three dimensions for cells to be clustered in biologically meaningful ways. Subsequently, gene expression is frequently mapped onto these plots to show the distribution of gene expression across the plots, for instance to distinguish cell types. However, plotting each cell with only a single color leads to repetitive and unintuitive representations. Here, we present PieParty, which allows scRNA-seq data to be plotted such that every cell is represented as a pie chart, and every slice in the pie charts corresponds to the gene expression of a single gene. This allows for the simultaneous visualization of the expression of multiple genes and gene networks. The resulting figures are information dense, space efficient, and highly intuitive. PieParty is publicly available on GitHub at https://github.com/harbourlab/PieParty.
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Perfilación de la Expresión Génica/métodos , Análisis de Secuencia de ARN/métodos , Análisis de la Célula Individual/métodos , Algoritmos , Secuencia de Bases/genética , Expresión Génica/genética , Redes Reguladoras de Genes/genética , Humanos , Procesamiento de Imagen Asistido por Computador/métodos , Programas Informáticos , Transcriptoma/genética , Secuenciación del Exoma/métodosRESUMEN
INTRODUCTION: Gene expression profiling (GEP) is widely used for prognostication in patients with uveal melanoma (UM). Because biopsy tissue is limited, it is critical to obtain as much genomic information as possible from each sample. Combined application of both GEP and next-generation sequencing (NGS) allows for analysis of RNA and DNA from a single biopsy sample, offers additional prognostic information, and can potentially inform therapy selection. This study evaluated the analytical performance of a targeted custom NGS panel for mutational profiling of 7 genes commonly mutated in UM. METHODS: One hundred five primary UM tumors were analyzed, including 37 formalin-fixed paraffin-embedded (FFPE) and 68 fine-needle aspiration biopsy specimens. Sequencing was performed on the Ion GeneStudio S5 platform to an average read depth of >500X per region of interest. RESULTS: The 7-gene panel achieved a positive percent agreement of 100% for detection of both single-nucleotide variants and insertions/deletions, with a technical positive predictive value of 98.8% and 100%, respectively. Intra-assay and inter-assay concordance studies confirmed the assay's reproducibility and repeatability. DISCUSSION/CONCLUSION: The 7-gene panel is a robust, highly accurate NGS test that can be successfully performed, along with GEP, from a single small-gauge needle biopsy sample or FFPE specimen.
RESUMEN
Uveal melanoma (UM) is the most common cancer of the eye and leads to metastatic death in up to half of patients. Genomic prognostic biomarkers play an important role in clinical management in UM. However, research has been conducted almost exclusively in patients of European descent, such that the association between genetic admixture and prognostic biomarkers is unknown. In this study, we compiled 1381 control genomes from West African, European, East Asian, and Native American individuals, assembled a bioinformatic pipeline for assessing global and local ancestry, and performed an initial pilot study of 141 UM patients from our international referral center that manages many admixed individuals. Global and local estimates were associated with genomic prognostic determinants. Expression quantitative trait loci (eQTL) analysis was performed on variants found in segments. Globally, after correction for multiple testing, no prognostic variable was significantly enriched in a given ancestral group. However, there was a trend suggesting an increased proportion of European ancestry associated with expression of the PRAME oncogene (q = 0.06). Locally enriched European haplotypes were associated with the poor prognosis class 2 gene expression profile and with genes involved in immune regulation (q = 4.7 × 10-11). These findings reveal potential influences of genetic ancestry on prognostic variables, implicate immune genes in prognostic differences based on ancestry, and provide a basis for future studies of admixed patients with UM using rigorous genetic ancestry methodology.
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Uveal melanoma (UM) is a highly metastatic cancer that, in contrast to cutaneous melanoma, is largely unresponsive to checkpoint immunotherapy. Here, we interrogate the tumor microenvironment at single-cell resolution using scRNA-seq of 59,915 tumor and non-neoplastic cells from 8 primary and 3 metastatic samples. Tumor cells reveal novel subclonal genomic complexity and transcriptional states. Tumor-infiltrating immune cells comprise a previously unrecognized diversity of cell types, including CD8+ T cells predominantly expressing the checkpoint marker LAG3, rather than PD1 or CTLA4. V(D)J analysis shows clonally expanded T cells, indicating that they are capable of mounting an immune response. An indolent liver metastasis from a class 1B UM is infiltrated with clonally expanded plasma cells, indicative of antibody-mediated immunity. This complex ecosystem of tumor and immune cells provides new insights into UM biology, and LAG3 is identified as a potential candidate for immune checkpoint blockade in patients with high risk UM.
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Melanoma/genética , Análisis de la Célula Individual , Neoplasias de la Úvea/genética , Línea Celular Tumoral , Análisis por Conglomerados , Variaciones en el Número de Copia de ADN/genética , Humanos , Melanoma/inmunología , Melanoma/patología , Metástasis de la Neoplasia , Análisis de Secuencia de ARN , Procesos Estocásticos , Transcripción Genética , Microambiente Tumoral/inmunología , Neoplasias de la Úvea/inmunología , Neoplasias de la Úvea/patología , Recombinación V(D)J/genéticaRESUMEN
BACKGROUND AND OBJECTIVE: To describe the presentation and the authors' evolving management strategy for intraocular dissemination of uveal melanoma cells following radiotherapy during the past decade. PATIENTS AND METHODS: Patients with uveal melanoma who developed intraocular dissemination of pigmented cells following radiotherapy. Histopathology was available in two cases. RESULTS: Four patients underwent treatment for progressive intraocular dissemination of uveal melanoma cells at 9 to 41 months following I-125 plaque radiotherapy (three patients) or proton beam radiotherapy (one patient). Treatments included primary enucleation (one patient), vitrectomy followed later by enucleation (one patient), and vitrectomy followed by intravitreal chemotherapy (two patients). Enucleated eyes demonstrated diffuse invasion of intraocular tissues by viable melanoma cells. No patient has developed systemic metastasis to date. CONCLUSIONS: Intraocular dissemination of pigmented cells following radiotherapy for uveal melanoma should raise suspicion for viable invasive melanoma cells. Prompt vitrectomy with intravitreal chemotherapy can be effective in avoiding enucleation in selected cases. [Ophthalmic Surg Lasers Imaging Retina. 2019;50:573-579.].
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Braquiterapia/efectos adversos , Neoplasias de la Coroides/radioterapia , Radioisótopos de Yodo/efectos adversos , Melanoma/radioterapia , Neoplasias de la Retina/patología , Cuerpo Vítreo/patología , Anciano , Neoplasias de la Coroides/patología , Oftalmopatías/patología , Enucleación del Ojo , Femenino , Humanos , Masculino , Melanoma/patología , Persona de Mediana Edad , Invasividad Neoplásica , Estudios RetrospectivosRESUMEN
Importance: There has been speculation on the pathogenesis of unilateral multifocal uveal melanoma, but there remains no convincing explanation. Genetic analysis suggests that unilateral multifocal uveal melanoma may represent intraocular metastasis with increased risk of systemic metastasis. Objective: To evaluate the pathogenesis of unilateral multifocal uveal melanoma. Design, Setting, and Participants: This clinical case series was conducted in tertiary academic ocular oncology referral centers and included patients with unilateral multifocal uveal melanoma. Main Outcomes and Measures: Gene expression and mutation profiling of tumor samples. Results: Four patients (all male; age range, 54-77 years) who were diagnosed with uveal melanoma were treated with plaque brachytherapy, and subsequently developed a second discrete uveal melanoma in the same eye were included. None demonstrated ocular or oculodermal melanocytosis. All 8 tumors available for analysis exhibited class 2 gene expression profiles. In all 4 cases, the initial and subsequent tumors were available for targeted DNA sequencing and identical driver mutations were present in both tumors. Data were collected from September 2015 to August 2018. Conclusions and Relevance: Unilateral multifocal uveal melanoma in the absence of ocular melanocytosis appears to occur preferentially in tumors with the class 2 gene expression profile and a BRCA1-associated protein 1 gene (BAP1) mutation. The presence of identical BAP1 mutations in multiple tumors in the same eye in the absence of a germline BAP1 mutation suggests intraocular metastasis rather than independent primary tumors. These findings indicate that the first site of metastasis can be within the eye itself and suggest that patients with unilateral multifocal uveal melanoma may be at increased risk of systemic metastasis.
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Subunidades alfa de la Proteína de Unión al GTP Gq-G11/genética , Subunidades alfa de la Proteína de Unión al GTP/genética , Mutación de Línea Germinal/genética , Melanoma/patología , Melanosis/patología , Neoplasias Primarias Secundarias/patología , Proteínas Supresoras de Tumor/genética , Ubiquitina Tiolesterasa/genética , Neoplasias de la Úvea/patología , Anciano , Biopsia con Aguja Fina , Braquiterapia , Enucleación del Ojo , Humanos , Radioisótopos de Yodo/uso terapéutico , Masculino , Melanoma/genética , Melanoma/radioterapia , Persona de Mediana Edad , Neoplasias Primarias Secundarias/genética , Neoplasias Primarias Secundarias/radioterapia , Estudios Retrospectivos , Neoplasias de la Úvea/genética , Neoplasias de la Úvea/radioterapiaRESUMEN
PURPOSE: The strong association between BAP1 mutations and metastasizing Class 2 uveal melanoma (UM) suggests that epigenetic alterations may play a significant role in tumor progression. Thus, we characterized the impact of BAP1 loss on the DNA methylome in UM.Experimental Design: Global DNA methylation was analyzed in 47 Class 1 and 45 Class 2 primary UMs and in UM cells engineered to inducibly deplete BAP1. RNA-Seq was analyzed in 80 UM samples and engineered UM cells. RESULTS: Hypermethylation on chromosome 3 correlated with downregulated gene expression at several loci, including 3p21, where BAP1 is located. Gene set analysis of hypermethylated and downregulated genes identified axon guidance and melanogenesis as deregulated pathways, with several of these genes located on chromosome 3. A novel hypermethylated site within the BAP1 locus was found in all Class 2 tumors, suggesting that BAP1 itself is epigenetically regulated. Highly differentially methylated probes were orthogonally validated using bisulfite sequencing, and they successfully distinguished Class 1 and Class 2 tumors in 100% of cases. In functional validation experiments, BAP1 knockdown in UM cells induced methylomic repatterning similar to UM tumors, enriched for genes involved in axon guidance, melanogenesis, and development. CONCLUSIONS: This study, coupled with previous work, suggests that the initial event in the divergence of Class 2 UM from Class 1 UM is loss of one copy of chromosome 3, followed by mutation of BAP1 on the remaining copy of chromosome 3, leading to the methylomic repatterning profile characteristic of Class 2 UMs.
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Metilación de ADN , Silenciador del Gen , Melanoma/genética , Melanoma/patología , Proteínas Supresoras de Tumor/genética , Ubiquitina Tiolesterasa/genética , Neoplasias de la Úvea/genética , Neoplasias de la Úvea/patología , Mapeo Cromosómico , Biología Computacional/métodos , Análisis Mutacional de ADN , Bases de Datos de Ácidos Nucleicos , Progresión de la Enfermedad , Epigénesis Genética , Epigenoma , Perfilación de la Expresión Génica , Técnicas de Silenciamiento del Gen , Humanos , Mutación , Clasificación del TumorRESUMEN
Ocular melanocytosis is the most important predisposing condition for the eye cancer uveal melanoma (UM). Here, we present a patient who developed UM arising within ocular melanocytosis who was treated with enucleation (eye removal), which provided an invaluable opportunity to interrogate both the UM and adjacent uveal tissue containing the melanocytosis using whole-exome and deep-targeted sequencing. This analysis revealed a clonal PLCB4 mutation in the melanocytosis, confirming that this is indeed a neoplastic condition and explaining why it predisposes to UM. This mutation was present in 100% of analyzed UM cells, indicating that a PLCB4-mutant cell gave rise to the UM. The earliest aberrations specific to the tumor were loss of Chromosomes 1p, 3, and 9p, which were present in virtually all tumor cells. A mutation in BAP1 arose later on the other copy of Chromosome 3 in a tumor subclone, followed by a gain of Chromosome 8q. These findings provide a mechanistic explanation for the well-known clinical association between ocular melanocytosis and UM by showing that this predisposing condition introduces the first "hit" and thereby increases the stochastic likelihood of acquiring further aberrations leading to UM.
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Melanoma/genética , Fosfolipasa C beta/genética , Neoplasias de la Úvea/genética , Exoma , Ojo/metabolismo , Femenino , Predisposición Genética a la Enfermedad/genética , Genómica , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Melanosis/genética , Persona de Mediana Edad , Mutación , Fosfolipasa C beta/metabolismo , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismo , Ubiquitina Tiolesterasa/genética , Ubiquitina Tiolesterasa/metabolismo , Secuenciación del ExomaRESUMEN
PURPOSE: To compare the prognostic accuracy of gene expression profiling (GEP) combined with PRAME status vs the clinical Tumor-Node-Metastasis (TNM) staging in patients with uveal melanoma (UM). DESIGN: Retrospective cohort study. METHODS: The study included 240 consecutive patients with UM. Tumors were assessed for GEP status (Class 1 or Class 2) using a validated 15-gene assay and PRAME expression status using quantitative polymerase chain reaction. TNM staging was according to the American Joint Committee on Cancer 8th edition. Statistical analysis included univariate and multivariate Cox proportional hazard models. Metastasis was the primary endpoint. RESULTS: GEP was Class 1 in 128 (53.3%) cases and Class 2 in 112 (46.7%) cases. PRAME status was negative in 157 (65.4%) cases and positive in 83 (34.6%) cases. TNM was stage I in 26 (10.8%) cases, IIA in 67 (27.9%) cases, IIB in 50 (20.8%) cases, IIIA in 59 (24.6%) cases, and IIIB in 38 (15.8%) cases. Metastatic disease was detected in 59 (24.6%) cases after median follow-up of 29 months (mean 42 months; range 1-195 months). Variables associated with metastasis included (in order of decreasing significance): GEP class (P = 1.5 × 10-8), largest basal tumor diameter (P = 2.5 × 10-6), PRAME status (P = 2.6 × 10-6), and TNM stage (P = 3.7 × 10-6). The prognostic accuracy of an optimized 3-category GEP/PRAME model (P = 8.6 × 10-14) was superior to an optimized TNM model (P = 1.3 × 10-5). CONCLUSIONS: In UM, molecular prognostic testing using GEP and PRAME provides prognostic accuracy that is superior to TNM staging.
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Antígenos de Neoplasias/genética , Perfilación de la Expresión Génica , Metástasis Linfática/patología , Melanoma/diagnóstico , Melanoma/genética , Neoplasias de la Úvea/diagnóstico , Neoplasias de la Úvea/genética , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Biomarcadores de Tumor/genética , Biopsia con Aguja Fina , Braquiterapia , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Radioisótopos de Yodo/uso terapéutico , Masculino , Melanoma/radioterapia , Persona de Mediana Edad , Estadificación de Neoplasias , Pronóstico , Modelos de Riesgos Proporcionales , Reacción en Cadena en Tiempo Real de la Polimerasa , Estudios Retrospectivos , Neoplasias de la Úvea/radioterapiaRESUMEN
Cancer is thought to arise through the accumulation of genomic aberrations evolving under Darwinian selection. However, it remains unclear when the aberrations associated with metastasis emerge during tumor evolution. Uveal melanoma (UM) is the most common primary eye cancer and frequently leads to metastatic death, which is strongly linked to BAP1 mutations. Accordingly, UM is ideally suited for studying the clonal evolution of metastatic competence. Here we analyze sequencing data from 151 primary UM samples using a customized bioinformatic pipeline, to improve detection of BAP1 mutations and infer the clonal relationships among genomic aberrations. Strikingly, we find BAP1 mutations and other canonical genomic aberrations usually arise in an early punctuated burst, followed by neutral evolution extending to the time of clinical detection. This implies that the metastatic proclivity of UM is "set in stone" early in tumor evolution and may explain why advances in primary treatment have not improved survival.
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Melanoma/genética , Proteínas Supresoras de Tumor/genética , Ubiquitina Tiolesterasa/genética , Neoplasias de la Úvea/genética , Análisis por Conglomerados , Variaciones en el Número de Copia de ADN , Metilación de ADN , Evolución Molecular , Humanos , Mutación , Secuenciación del ExomaRESUMEN
PURPOSE: Uveal melanoma (UM) can be classified by gene expression profiling (GEP) into Class 1 (low metastatic risk) and Class 2 (high metastatic risk), the latter being strongly associated with mutational inactivation of the tumor suppressor BAP1. Nevertheless, a small percentage of Class 1 tumors give rise to metastatic disease. The purpose of this study was to identify biomarkers of metastasis in Class 1 tumors. EXPERIMENTAL DESIGN: A total of 389 consecutive patients with UM were assigned to Class 1 or Class 2 using a prospectively validated 12-gene prognostic classifier. Selected tumors were further analyzed using global GEP and single nucleotide polymorphism microarrays. PRAME (preferentially expressed antigen in melanoma) mRNA expression was analyzed in 64 Class 1 tumors by qPCR. RESULTS: Among Class 1 UMs, the most significant predictor of metastasis was PRAME mRNA expression (P = 0.0006). The 5-year actuarial rate of metastasis was 0% for Class1(PRAME-), 38% for Class1(PRAME+), and 71% for Class 2 tumors. Median metastasis-free survival for Class1(PRAME+) patients was 88 months, compared to 32 months for Class 2 patients. Findings were validated using three independent datasets, including one using disomy 3 to identify low-risk UM. Chromosome copy number changes associated with Class1(PRAME+) tumors included gain of 1q, 6p, 8q, and 9q and loss of 6q and 11q. PRAME expression was associated with larger tumor diameter (P = 0.05) and SF3B1 mutations (P = 0.003). CONCLUSIONS: PRAME is an independent prognostic biomarker in UM, which identifies increased metastatic risk in patients with Class 1 or disomy 3 tumors. This finding may further enhance the accuracy of prognostic testing and precision medicine for UM.
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Antígenos de Neoplasias/biosíntesis , Biomarcadores de Tumor/biosíntesis , Melanoma/genética , Metástasis de la Neoplasia/genética , Neoplasias de la Úvea/genética , Adulto , Anciano , Antígenos de Neoplasias/genética , Biomarcadores de Tumor/genética , Biopsia con Aguja Fina , Supervivencia sin Enfermedad , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Estimación de Kaplan-Meier , Masculino , Melanoma/clasificación , Melanoma/patología , Persona de Mediana Edad , Metástasis de la Neoplasia/patología , Fosfoproteínas/genética , Factores de Empalme de ARN/genética , Proteínas Supresoras de Tumor/genética , Ubiquitina Tiolesterasa/genética , Neoplasias de la Úvea/clasificación , Neoplasias de la Úvea/patologíaRESUMEN
IMPORTANCE: Frequent mutations have been described in the following 5 genes in uveal melanoma (UM): BAP1, EIF1AX, GNA11, GNAQ, and SF3B1. Understanding the prognostic significance of these mutations could facilitate their use in precision medicine. OBJECTIVE: To determine the associations between driver mutations, gene expression profile (GEP) classification, clinicopathologic features, and patient outcomes in UM. DESIGN, SETTING, AND PARTICIPANTS: Retrospective study of patients with UM treated by enucleation by a single ocular oncologist between November 1, 1998, and July 31, 2014. MAIN OUTCOMES AND MEASURES: Clinicopathologic features, patient outcomes, GEP classification (class 1 or class 2), and mutation status were recorded. RESULTS: The study cohort comprised 81 participants. Their mean age was 61.5 years, and 37% (30 of 81) were female. The GEP classification was class 1 in 35 of 81 (43%), class 2 in 42 of 81 (52%), and unknown in 4 of 81 (5%). BAP1 mutations were identified in 29 of 64 (45%), GNAQ mutations in 36 of 81 (44%), GNA11 mutations in 36 of 81 (44%), SF3B1 mutations in 19 of 81 (24%), and EIF1AX mutations in 14 of 81 (17%). Sixteen of the mutations in BAP1 and 6 of the mutations in EIF1AX were previously unreported in UM. GNAQ and GNA11 mutations were mutually exclusive. BAP1, SF3B1, and EIF1AX mutations were almost mutually exclusive with each other. Using multiple regression analysis, BAP1 mutations were associated with class 2 GEP and older patient. EIF1AX mutations were associated with class 1 GEP and the absence of ciliary body involvement. SF3B1 mutations were associated with younger patient age. GNAQ mutations were associated with the absence of ciliary body involvement and greater largest basal diameter. GNA11 mutations were not associated with any of the analyzed features. Using Cox proportional hazards modeling, class 2 GEP was the prognostic factor most strongly associated with metastasis (relative risk, 9.4; 95% CI, 3.1-28.5) and melanoma-specific mortality (relative risk, 15.7; 95% CI, 3.6-69.1) (P < .001 for both). After excluding GEP class, the presence of BAP1 mutations was the factor most strongly associated with metastasis (relative risk, 10.6; 95% CI, 3.4-33.5) and melanoma-specific mortality (relative risk, 9.0; 95% CI, 2.8-29.2) (P < .001 for both). CONCLUSIONS AND RELEVANCE: BAP1, SF3B1, and EIF1AX mutations occur during UM tumor progression in an almost mutually exclusive manner and are associated with different levels of metastatic risk. These mutations may have value as prognostic markers in UM.
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Factor 1 Eucariótico de Iniciación/genética , Regulación Neoplásica de la Expresión Génica/fisiología , Melanoma/genética , Mutación , Proteínas de Neoplasias/genética , Fosfoproteínas/genética , Factores de Empalme de ARN/genética , Proteínas Supresoras de Tumor/genética , Ubiquitina Tiolesterasa/genética , Neoplasias de la Úvea/genética , Análisis Mutacional de ADN , Femenino , Perfilación de la Expresión Génica , Humanos , Masculino , Melanoma/diagnóstico , Persona de Mediana Edad , Reacción en Cadena de la Polimerasa , Pronóstico , Estudios Retrospectivos , Neoplasias de la Úvea/diagnósticoRESUMEN
Interference with endothelial cell metabolism is a promising, yet unexploited strategy for angiogenesis inhibition. We reported that the glucose analogue 2-deoxy-D-glucose (2-DG) inhibits angiogenesis at significantly lower concentrations than those required for tumor cytotoxicity. Here, we found that hypersensitivity to 2-DG in endothelial cells is not associated with enhanced drug uptake compared with tumor cells, but with time-dependent, endothelial-selective inhibition of AKT and ERK phosphorylation. Downregulation of these critical survival pathways is shown to be due to 2-DG's interference with N-linked glycosylation, leading to alterations in VEGFR2 (and downstream signaling) as well as induction of endoplasmic reticulum (ER) stress, GSK3ß activation, and apoptosis. In vivo, periocular administration of 2-DG in LHBETATAG mice was associated with significant reduction of newly formed (CD105(+)) tumor capillaries, ER stress (GRP 78 expression), and endothelial apoptosis (TUNEL). These findings uniquely link N-linked glycosylation inhibition, ER stress, and ERK/AKT downregulation in endothelial cells, and provide a novel drug development strategy to overcome resistance mechanisms to currently available antiangiogenic agents.
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Inhibidores de la Angiogénesis/administración & dosificación , Desoxiglucosa/administración & dosificación , Estrés del Retículo Endoplásmico/efectos de los fármacos , Células Endoteliales/efectos de los fármacos , Glucógeno Sintasa Quinasa 3/metabolismo , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Inhibidores de la Angiogénesis/farmacología , Animales , Apoptosis , Línea Celular Tumoral , Desoxiglucosa/farmacología , Regulación hacia Abajo , Células Endoteliales/metabolismo , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Glucógeno Sintasa Quinasa 3 beta , Glicosilación/efectos de los fármacos , Células HT29 , Células Endoteliales de la Vena Umbilical Humana , Humanos , Ratones , Proteínas Proto-Oncogénicas c-akt/metabolismo , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismoRESUMEN
BACKGROUND: We previously identified PRAME as a biomarker for metastatic risk in Class 1 uveal melanomas. In this study, we sought to define a threshold value for positive PRAME expression (PRAME+) in a large dataset, identify factors associated with PRAME expression, evaluate the prognostic value of PRAME in Class 2 uveal melanomas, and determine whether PRAME expression is associated with aberrant hypomethylation of the PRAME promoter. RESULTS: Among 678 samples analyzed by qPCR, 498 (73.5%) were PRAME- and 180 (26.5%) were PRAME+. Class 1 tumors were more likely to be PRAME-, whereas Class 2 tumors were more likely to be PRAME+ (P < 0.0001). PRAME expression was associated with shorter time to metastasis and melanoma specific mortality in Class 2 tumors (P = 0.01 and P = 0.02, respectively). In Class 1 tumors, PRAME expression was directly associated with SF3B1 mutations (P < 0.0001) and inversely associated with EIF1AX mutations (P = 0.004). PRAME expression was strongly associated with hypomethylation at 12 CpG sites near the PRAME promoter. MATERIALS AND METHODS: Analyses included PRAME mRNA expression, Class 1 versus Class 2 status, chromosomal copy number, mutation status of BAP1, EIF1AX, GNA11, GNAQ and SF3B1, and genomic DNA methylation status. Analyses were performed on 555 de-identified samples from Castle Biosciences, 123 samples from our center, and 80 samples from the TCGA. CONCLUSIONS: PRAME is aberrantly hypomethylated and activated in Class 1 and Class 2 uveal melanomas and is associated with increased metastatic risk in both classes. Since PRAME has been successfully targeted for immunotherapy, it may prove to be a companion prognostic biomarker.