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BRCA-mutant cancers often develop therapeutic resistance through several mechanisms. Here, we report a case of pathogenic germline BRCA2-driven breast cancer monitored for disease progression and acquired resistance using longitudinal multi-tissue genomic testing. Briefly, genomic testing was performed throughout the course of disease on tumor tissue from multiple sites, circulating tumor DNA from blood plasma, and matched normal tissue. Genomic analyses identified actionable variants for targeted therapies, as well as emerging resistance mutations over time. Two unique BRCA2 somatic alterations (p.N255fs and p.D252fs) were identified upon resistance to PARP inhibitor and platinum treatment, respectively. Both alterations restored the open reading frame of the original germline alteration, likely accounting for acquired resistance. This case exemplifies the evolution of multiple subclonal BRCA reversion alterations over time and demonstrates the value of longitudinal multi-tissue genomic testing for monitoring disease progression, predicting measures of response, and evaluating treatment outcomes in oncology patients.
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We developed and clinically validated a hybrid capture next generation sequencing assay to detect somatic alterations and microsatellite instability in solid tumors and hematologic malignancies. This targeted oncology assay utilizes tumor-normal matched samples for highly accurate somatic alteration calling and whole transcriptome RNA sequencing for unbiased identification of gene fusion events. The assay was validated with a combination of clinical specimens and cell lines, and recorded a sensitivity of 99.1% for single nucleotide variants, 98.1% for indels, 99.9% for gene rearrangements, 98.4% for copy number variations, and 99.9% for microsatellite instability detection. This assay presents a wide array of data for clinical management and clinical trial enrollment while conserving limited tissue.
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RNA splicing factors are essential for the viability of all eukaryotic cells; however, in metazoans some cell types are exquisitely sensitive to disruption of splicing factors. Neuronal cells represent one such cell type, and defects in RNA splicing factors can lead to neurodegenerative diseases. The basis for this tissue selectivity is not well understood owing to difficulties in analyzing the consequences of splicing factor defects in whole-animal systems. Here, we use zebrafish mutants to show that loss of spliceosomal components, including splicing factor 3b, subunit 1 (sf3b1), causes increased DNA double-strand breaks and apoptosis in embryonic neurons. Moreover, these mutants show a concomitant accumulation of R-loops, which are non-canonical nucleic acid structures that promote genomic instability. Dampening R-loop formation by conditional induction of ribonuclease H1 in sf3b1 mutants reduced neuronal DNA damage and apoptosis. These findings show that splicing factor dysfunction leads to R-loop accumulation and DNA damage that sensitizes embryonic neurons to apoptosis. Our results suggest that diseases associated with splicing factor mutations could be susceptible to treatments that modulate R-loop levels.
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Apoptosis , Citoprotección , Daño del ADN , Neuronas/citología , Neuronas/metabolismo , Conformación de Ácido Nucleico , Empalmosomas/metabolismo , Pez Cebra/genética , Animales , Apoptosis/efectos de la radiación , Citoprotección/efectos de la radiación , Roturas del ADN de Doble Cadena , Genes Esenciales , Mutación/genética , Neuronas/efectos de la radiación , Empalme del ARN/genética , Empalme del ARN/efectos de la radiación , Tolerancia a Radiación/genética , Tolerancia a Radiación/efectos de la radiación , Radiación Ionizante , Proteína p53 Supresora de Tumor/metabolismo , Pez Cebra/embriología , Proteínas de Pez Cebra/metabolismoRESUMEN
AIMS: Genetic abnormalities, including copy number variants (CNV), copy number neutral loss of heterozygosity (CN-LOH) and gene mutations, underlie the pathogenesis of myeloid malignancies and serve as important diagnostic, prognostic and/or therapeutic markers. Currently, multiple testing strategies are required for comprehensive genetic testing in myeloid malignancies. The aim of this proof-of-principle study was to investigate the feasibility of combining detection of genome-wide large CNVs, CN-LOH and targeted gene mutations into a single assay using next-generation sequencing (NGS). METHODS: For genome-wide CNV detection, we designed a single nucleotide polymorphism (SNP) sequencing backbone with 22 762 SNP regions evenly distributed across the entire genome. For targeted mutation detection, 62 frequently mutated genes in myeloid malignancies were targeted. We combined this SNP sequencing backbone with a targeted mutation panel, and sequenced 9 healthy individuals and 16 patients with myeloid malignancies using NGS. RESULTS: We detected 52 somatic CNVs, 11 instances of CN-LOH and 39 oncogenic mutations in the 16 patients with myeloid malignancies, and none in the 9 healthy individuals. All CNVs and CN-LOH were confirmed by SNP microarray analysis. CONCLUSIONS: We describe a genome-wide SNP sequencing backbone which allows for sensitive detection of genome-wide CNVs and CN-LOH using NGS. This proof-of-principle study has demonstrated that this strategy can provide more comprehensive genetic profiling for patients with myeloid malignancies using a single assay.
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Variaciones en el Número de Copia de ADN/genética , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Trastornos Mieloproliferativos/genética , Femenino , Humanos , Masculino , Polimorfismo de Nucleótido Simple/genéticaRESUMEN
NPM1 insertion mutations represent a common recurrent genetic abnormality in acute myeloid leukemia (AML) patients. The frequency of these mutations varies from approximately 30% overall up to 50% in patients with a normal karyotype. Several recent studies have exploited advances in massively parallel sequencing technology to shed light on the complex genomic landscape of AML. We hypothesize that variant allele fraction (VAF) data derived from massively parallel sequencing studies may provide further insights into the clonal architecture and pathogenesis of NPM1-driven leukemogenesis. Diagnostic peripheral blood or bone marrow samples from NPM1-mutated AML patients (n=120) were subjected to targeted sequencing using a panel of fifty-seven genes known to be commonly mutated in myeloid malignancies. NPM1 mutations were always accompanied by additional mutations and NPM1 had the highest VAF in only one case. Nearly all NPM1-mutated AML patients showed concurrent mutations in genes involved in regulation of DNA methylation (DNMT3A, TET2, IDH1, IDH2), RNA splicing (SRSF2, SF3B1), or in the cohesin complex (RAD21, SMC1A, SMC3, STAG2). Mutations in these genes had higher median VAFs that were higher (40% or greater) than the co-existing NPM1 mutations (median VAF 16.8%). Mutations associated with cell signaling pathways (FLT3, NRAS, and PTPN11) are also frequently encountered in NPM1-mutated AML cases, but had relatively low VAFs (7.0-11.9%). No cases of NPM1-mutated AML with a concurrent IDH2R172 mutation were observed, suggesting that these variants are mutually exclusive. Overall, these data suggest that NPM1 mutations are a secondary or late event in the pathogenesis of AML and are preceded by founder mutations in genes that may be associated with recently described preclinical states such as clonal hematopoiesis of indeterminate potential or clonal cytopenias of undetermined significance.
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Leucemia Mieloide Aguda/genética , Mutación , Proteínas Nucleares/genética , Proteínas de Ciclo Celular/genética , Proteínas Cromosómicas no Histona/genética , Metilación de ADN/genética , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Nucleofosmina , Empalme del ARN/genética , CohesinasRESUMEN
Simian virus 40 (SV40) large T antigen (SVT) interferes with normal cell regulation and thus has been used to identify cellular components controlling proliferation and homeostasis. We have previously shown that SVT-mediated transformation requires interaction with the histone acetyltransferases (HATs) CBP/p300 and now report that the ectopic expression of SVT in several cell types in vivo and in vitro results in a significant increase in the steady-state levels of CBP/p300. Furthermore, SVT-expressing cells contain higher levels of acetylated CBP/p300, a modification that has been linked to increased HAT activity. Concomitantly, the acetylation levels of histone residues H3K56 and H4K12 are markedly increased in SVT-expressing cells. Other polyomavirus-encoded large T antigens also increase the levels of CBP/p300 and sustain a rise in the acetylation levels of H3K56 and H4K12. SVT does not affect the transcription of CBP/p300, but rather, alters their overall levels through increasing the loading of CBP/p300 mRNAs onto polysomes. Two distinct regions within SVT, one located in the amino terminus and one in the carboxy terminus, can independently alter both the levels of CBP/p300 and the loading of CBP/p300 transcripts onto polysomes. Within the amino-terminal fragment, a functional J domain is necessary for increasing CBP/p300 and specific histone acetylation levels, as well as for immortalizing primary cells. These studies uncover the action of polyomavirus T antigens on cellular CBP/p300 and suggest that additional mechanisms are used by T antigens to induce cell immortalization and transformation.
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Antígenos Transformadores de Poliomavirus/química , Antígenos Transformadores de Poliomavirus/metabolismo , Proteína de Unión a CREB/metabolismo , Transformación Celular Viral , Proteína p300 Asociada a E1A/metabolismo , Histonas/metabolismo , Infecciones por Polyomavirus/metabolismo , Virus 40 de los Simios/fisiología , Acetilación , Secuencias de Aminoácidos , Animales , Antígenos Transformadores de Poliomavirus/genética , Proteína de Unión a CREB/genética , Células Cultivadas , Proteína p300 Asociada a E1A/genética , Fibroblastos/metabolismo , Fibroblastos/virología , Histonas/química , Histonas/genética , Humanos , Infecciones por Polyomavirus/enzimología , Infecciones por Polyomavirus/genética , Infecciones por Polyomavirus/virología , Virus 40 de los Simios/química , Virus 40 de los Simios/genética , Infecciones Tumorales por Virus/enzimología , Infecciones Tumorales por Virus/genética , Infecciones Tumorales por Virus/metabolismo , Infecciones Tumorales por Virus/virologíaRESUMEN
Whole-mount immunofluorescence to detect activated Caspase 3 (Casp3 assay) is useful to identify cells undergoing either intrinsic or extrinsic apoptosis in zebrafish embryos. The whole-mount analysis provides spatial information in regard to tissue specificity of apoptosing cells, although sectioning and/or colabeling is ultimately required to pinpoint the exact cell types undergoing apoptosis. The whole-mount Casp3 assay is optimized for analysis of fixed embryos between the 4-cell stage and 32 hr-post-fertilization and is useful for a number of applications, including analysis of zebrafish mutants and morphants, overexpression of mutant and wild-type mRNAs, and exposure to chemicals. Compared to acridine orange staining, which can identify apoptotic cells in live embryos in a matter of hours, Casp3 and TUNEL assays take considerably longer to complete (2-4 days). However, because of the dynamic nature of apoptotic cell formation and clearance, analysis of fixed embryos ensures accurate comparison of apoptotic cells across multiple samples at specific time points. We have also found the Casp3 assay to be superior to analysis of apoptotic cells by the whole-mount TUNEL assay in regard to cost and reliability. Overall, the Casp3 assay represents a robust, highly reproducible assay in which to analyze apoptotic cells in early zebrafish embryos.
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Apoptosis/fisiología , Caspasa 3/metabolismo , Técnicas de Cultivo de Embriones/métodos , Técnica del Anticuerpo Fluorescente/métodos , Pez Cebra/embriología , Animales , Caspasa 3/análisis , Embrión no Mamífero/anatomía & histología , Embrión no Mamífero/citología , Activación Enzimática , Pez Cebra/metabolismoRESUMEN
DNA double-strand breaks (DSBs) represent one of the most deleterious forms of DNA damage to a cell. In cancer therapy, induction of cell death by DNA DSBs by ionizing radiation (IR) and certain chemotherapies is thought to mediate the successful elimination of cancer cells. However, cancer cells often evolve to evade the cytotoxicity induced by DNA DSBs, thereby forming the basis for treatment resistance. As such, a better understanding of the DSB DNA damage response (DSB-DDR) pathway will facilitate the design of more effective strategies to overcome chemo- and radioresistance. To identify novel mechanisms that protect cells from the cytotoxic effects of DNA DSBs, we performed a forward genetic screen in zebrafish for recessive mutations that enhance the IR-induced apoptotic response. Here, we describe radiosensitizing mutation 7 (rs7), which causes a severe sensitivity of zebrafish embryonic neurons to IR-induced apoptosis and is required for the proper development of the central nervous system. The rs7 mutation disrupts the coding sequence of ccdc94, a highly conserved gene that has no previous links to the DSB-DDR pathway. We demonstrate that Ccdc94 is a functional member of the Prp19 complex and that genetic knockdown of core members of this complex causes increased sensitivity to IR-induced apoptosis. We further show that Ccdc94 and the Prp19 complex protect cells from IR-induced apoptosis by repressing the expression of p53 mRNA. In summary, we have identified a new gene regulating a dosage-sensitive response to DNA DSBs during embryonic development. Future studies in human cancer cells will determine whether pharmacological inactivation of CCDC94 reduces the threshold of the cancer cell apoptotic response.