RESUMEN
Accurate measurement of clonal genotypes, mutational processes, and replication states from individual tumor-cell genomes will facilitate improved understanding of tumor evolution. We have developed DLP+, a scalable single-cell whole-genome sequencing platform implemented using commodity instruments, image-based object recognition, and open source computational methods. Using DLP+, we have generated a resource of 51,926 single-cell genomes and matched cell images from diverse cell types including cell lines, xenografts, and diagnostic samples with limited material. From this resource we have defined variation in mitotic mis-segregation rates across tissue types and genotypes. Analysis of matched genomic and image measurements revealed correlations between cellular morphology and genome ploidy states. Aggregation of cells sharing copy number profiles allowed for calculation of single-nucleotide resolution clonal genotypes and inference of clonal phylogenies and avoided the limitations of bulk deconvolution. Finally, joint analysis over the above features defined clone-specific chromosomal aneuploidy in polyclonal populations.
Asunto(s)
Replicación del ADN/genética , Genoma Humano , Secuenciación de Nucleótidos de Alto Rendimiento , Análisis de la Célula Individual , Aneuploidia , Animales , Ciclo Celular/genética , Línea Celular Tumoral , Forma de la Célula , Supervivencia Celular , Cromosomas Humanos/genética , Células Clonales , Elementos Transponibles de ADN/genética , Diploidia , Femenino , Genotipo , Humanos , Masculino , Ratones , Mutación/genética , Filogenia , Polimorfismo de Nucleótido Simple/genéticaRESUMEN
While the preponderance of morbidity and mortality in medulloblastoma patients are due to metastatic disease, most research focuses on the primary tumor due to a dearth of metastatic tissue samples and model systems. Medulloblastoma metastases are found almost exclusively on the leptomeningeal surface of the brain and spinal cord; dissemination is therefore thought to occur through shedding of primary tumor cells into the cerebrospinal fluid followed by distal re-implantation on the leptomeninges. We present evidence for medulloblastoma circulating tumor cells (CTCs) in therapy-naive patients and demonstrate in vivo, through flank xenografting and parabiosis, that medulloblastoma CTCs can spread through the blood to the leptomeningeal space to form leptomeningeal metastases. Medulloblastoma leptomeningeal metastases express high levels of the chemokine CCL2, and expression of CCL2 in medulloblastoma in vivo is sufficient to drive leptomeningeal dissemination. Hematogenous dissemination of medulloblastoma offers a new opportunity to diagnose and treat lethal disseminated medulloblastoma.
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Meduloblastoma/irrigación sanguínea , Meduloblastoma/patología , Neoplasias Meníngeas/irrigación sanguínea , Neoplasias Meníngeas/secundario , Aloinjertos , Animales , Línea Celular Tumoral , Quimiocina CCL2/metabolismo , Cromosomas Humanos Par 10/genética , Femenino , Humanos , Masculino , Meduloblastoma/genética , Ratones SCID , Células Neoplásicas Circulantes , ParabiosisRESUMEN
Progress in defining genomic fitness landscapes in cancer, especially those defined by copy number alterations (CNAs), has been impeded by lack of time-series single-cell sampling of polyclonal populations and temporal statistical models1-7. Here we generated 42,000 genomes from multi-year time-series single-cell whole-genome sequencing of breast epithelium and primary triple-negative breast cancer (TNBC) patient-derived xenografts (PDXs), revealing the nature of CNA-defined clonal fitness dynamics induced by TP53 mutation and cisplatin chemotherapy. Using a new Wright-Fisher population genetics model8,9 to infer clonal fitness, we found that TP53 mutation alters the fitness landscape, reproducibly distributing fitness over a larger number of clones associated with distinct CNAs. Furthermore, in TNBC PDX models with mutated TP53, inferred fitness coefficients from CNA-based genotypes accurately forecast experimentally enforced clonal competition dynamics. Drug treatment in three long-term serially passaged TNBC PDXs resulted in cisplatin-resistant clones emerging from low-fitness phylogenetic lineages in the untreated setting. Conversely, high-fitness clones from treatment-naive controls were eradicated, signalling an inversion of the fitness landscape. Finally, upon release of drug, selection pressure dynamics were reversed, indicating a fitness cost of treatment resistance. Together, our findings define clonal fitness linked to both CNA and therapeutic resistance in polyclonal tumours.
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Variaciones en el Número de Copia de ADN , Resistencia a Antineoplásicos , Neoplasias de la Mama Triple Negativas/genética , Animales , Línea Celular Tumoral , Cisplatino/farmacología , Células Clonales/patología , Femenino , Aptitud Genética , Humanos , Ratones , Modelos Estadísticos , Trasplante de Neoplasias , Proteína p53 Supresora de Tumor/genética , Secuenciación Completa del GenomaRESUMEN
Follicular lymphoma (FL) accounts for â¼20% of all new lymphoma cases. Increases in cytological grade are a feature of the clinical progression of this malignancy, and eventual histologic transformation (HT) to the aggressive diffuse large B-cell lymphoma (DLBCL) occurs in up to 15% of patients. Clinical or genetic features to predict the risk and timing of HT have not been described comprehensively. In this study, we analyzed whole-genome sequencing data from 423 patients to compare the protein coding and noncoding mutation landscapes of untransformed FL, transformed FL, and de novo DLBCL. This revealed 2 genetically distinct subgroups of FL, which we have named DLBCL-like (dFL) and constrained FL (cFL). Each subgroup has distinguishing mutational patterns, aberrant somatic hypermutation rates, and biological and clinical characteristics. We implemented a machine learning-derived classification approach to stratify patients with FL into cFL and dFL subgroups based on their genomic features. Using separate validation cohorts, we demonstrate that cFL status, whether assigned with this full classifier or a single-gene approximation, is associated with a reduced rate of HT. This implies distinct biological features of cFL that constrain its evolution, and we highlight the potential for this classification to predict HT from genetic features present at diagnosis.
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Linfoma Folicular , Linfoma de Células B Grandes Difuso , Humanos , Linfoma Folicular/patología , Mutación , Linfoma de Células B Grandes Difuso/genética , Linfoma de Células B Grandes Difuso/patologíaRESUMEN
Study of the origin and development of cerebellar tumours has been hampered by the complexity and heterogeneity of cerebellar cells that change over the course of development. Here we use single-cell transcriptomics to study more than 60,000 cells from the developing mouse cerebellum and show that different molecular subgroups of childhood cerebellar tumours mirror the transcription of cells from distinct, temporally restricted cerebellar lineages. The Sonic Hedgehog medulloblastoma subgroup transcriptionally mirrors the granule cell hierarchy as expected, while group 3 medulloblastoma resembles Nestin+ stem cells, group 4 medulloblastoma resembles unipolar brush cells, and PFA/PFB ependymoma and cerebellar pilocytic astrocytoma resemble the prenatal gliogenic progenitor cells. Furthermore, single-cell transcriptomics of human childhood cerebellar tumours demonstrates that many bulk tumours contain a mixed population of cells with divergent differentiation. Our data highlight cerebellar tumours as a disorder of early brain development and provide a proximate explanation for the peak incidence of cerebellar tumours in early childhood.
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Neoplasias Cerebelosas/genética , Neoplasias Cerebelosas/patología , Evolución Molecular , Feto/metabolismo , Regulación del Desarrollo de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Transcripción Genética , Animales , Neoplasias Cerebelosas/clasificación , Cerebelo/citología , Cerebelo/embriología , Cerebelo/metabolismo , Niño , Femenino , Feto/citología , Glioma/clasificación , Glioma/genética , Glioma/patología , Humanos , Meduloblastoma/clasificación , Meduloblastoma/genética , Meduloblastoma/patología , Ratones , Análisis de Secuencia de ARN , Análisis de la Célula Individual , Factores de Tiempo , Transcriptoma/genéticaRESUMEN
SUMMARY: Mapping genetic interactions and essentiality networks in human cell lines has been used to identify vulnerabilities of cells carrying specific genetic alterations and to associate novel functions to genes, respectively. In vitro and in vivo genetic screens to decipher these networks are resource-intensive, limiting the throughput of samples that can be analyzed. In this application note, we provide an R package we call Genetic inteRaction and EssenTiality neTwork mApper (GRETTA). GRETTA is an accessible tool for in silico genetic interaction screens and essentiality network analyses using publicly available data, requiring only basic R programming knowledge. AVAILABILITY AND IMPLEMENTATION: The R package, GRETTA, is licensed under GNU General Public License v3.0 and freely available at https://github.com/ytakemon/GRETTA and https://doi.org/10.5281/zenodo.6940757, with documentation and tutorial. A Singularity container is also available at https://cloud.sylabs.io/library/ytakemon/gretta/gretta.
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Programas Informáticos , Humanos , MutaciónRESUMEN
Mixed phenotype acute leukaemia (MPAL) is a high-risk subtype of leukaemia with myeloid and lymphoid features, limited genetic characterization, and a lack of consensus regarding appropriate therapy. Here we show that the two principal subtypes of MPAL, T/myeloid (T/M) and B/myeloid (B/M), are genetically distinct. Rearrangement of ZNF384 is common in B/M MPAL, and biallelic WT1 alterations are common in T/M MPAL, which shares genomic features with early T-cell precursor acute lymphoblastic leukaemia. We show that the intratumoral immunophenotypic heterogeneity characteristic of MPAL is independent of somatic genetic variation, that founding lesions arise in primitive haematopoietic progenitors, and that individual phenotypic subpopulations can reconstitute the immunophenotypic diversity in vivo. These findings indicate that the cell of origin and founding lesions, rather than an accumulation of distinct genomic alterations, prime tumour cells for lineage promiscuity. Moreover, these findings position MPAL in the spectrum of immature leukaemias and provide a genetically informed framework for future clinical trials of potential treatments for MPAL.
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Leucemia Bifenotípica Aguda/genética , Leucemia Bifenotípica Aguda/patología , Linaje de la Célula/genética , Análisis Mutacional de ADN , Femenino , Variación Genética/genética , Genoma Humano/genética , Genómica , Humanos , Inmunofenotipificación , Leucemia Bifenotípica Aguda/clasificación , Masculino , Modelos Genéticos , Mutación/genética , Células Madre Neoplásicas/inmunología , Células Madre Neoplásicas/metabolismo , Células Madre Neoplásicas/patología , Fenotipo , Transactivadores/genéticaRESUMEN
When the World Health Organization defined high-grade B-cell lymphoma with MYC and BCL2 and/or BCL6 rearrangements (HGBL-DH/TH) as a clinical category, rearrangements were the only structural variant (SV) incorporated. An "atypical double-hit" category has been proposed, encompassing tumors with concurrent MYC and BCL2 SVs other than cooccurring translocations (ie, copy number variations [CNVs]). Although the identification of a gene expression signature (DHITsig) shared among tumors harboring MYC and BCL2 rearrangements (HGBL-DH/TH-BCL2) has confirmed a common underlying biology, the biological implication of MYC and BCL2 CNVs requires further elucidation. We performed a comprehensive analysis of MYC and BCL2 SVs, as determined by fluorescent in situ hybridization (FISH), in a cohort of 802 de novo tumors with diffuse large B-cell lymphoma morphology. Although BCL2 CNVs were associated with increased expression, MYC CNVs were not. Furthermore, MYC and BCL2 CNVs, in the context of atypical double-hit, did not confer a similar gene expression profile as HGBL-DH/TH-BCL2. Finally, although MYC immunohistochemistry (IHC) has been proposed as a screening tool for FISH testing, 2 mechanisms were observed that uncoupled MYC rearrangement from IHC positivity: (1) low MYC messenger RNA expression; and (2) false-negative IHC staining mediated by a single-nucleotide polymorphism resulting in an asparagine-to-serine substitution at the 11th amino acid residue of MYC (MYC-N11S). Taken together, these results support the current exclusion of MYC and BCL2 CNVs from HGBL-DH/TH and highlight the ability of a molecular-based classification system to identify tumors with shared biology that FISH and IHC fail to fully capture.
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Linfoma de Células B Grandes Difuso/genética , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteínas Proto-Oncogénicas c-myc/genética , Adulto , Anciano , Anciano de 80 o más Años , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Inmunohistoquímica , Masculino , Persona de Mediana Edad , Polimorfismo de Nucleótido Simple , Proteínas Proto-Oncogénicas c-bcl-2/análisis , Proteínas Proto-Oncogénicas c-myc/análisis , Transcriptoma , Adulto JovenRESUMEN
Capicua (CIC)'s transcriptional repressor function is implicated in neurodevelopment and in oligodendroglioma (ODG) aetiology. However, CIC's role in these contexts remains obscure, primarily from our currently limited knowledge regarding its biological functions. Moreover, CIC mutations in ODG invariably co-occur with a neomorphic IDH1/2 mutation, yet the functional relationship between these two genetic events is unknown. Here, we analysed models derived from an E6/E7/hTERT-immortalized (i.e. p53- and RB-deficient) normal human astrocyte cell line. To examine the consequences of CIC loss, we compared transcriptomic and epigenomic profiles between CIC wild-type and knockout cell lines, with and without mutant IDH1 expression. Our analyses revealed dysregulation of neurodevelopmental genes in association with CIC loss. CIC ChIP-seq was also performed to expand upon the currently limited ensemble of known CIC target genes. Among the newly identified direct CIC target genes were EPHA2 and ID1, whose functions are linked to neurodevelopment and the tumourigenicity of in vivo glioma tumour models. NFIA, a known mediator of gliogenesis, was discovered to be uniquely overexpressed in CIC-knockout cells expressing mutant IDH1-R132H protein. These results identify neurodevelopment and specific genes within this context as candidate targets through which CIC alterations may contribute to the progression of IDH-mutant gliomas. © 2021 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd on behalf of The Pathological Society of Great Britain and Ireland.
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Astrocitos/enzimología , Epigenoma , Epigenómica , Perfilación de la Expresión Génica , Isocitrato Deshidrogenasa/genética , Mutación , Proteínas Represoras/genética , Transcriptoma , Astrocitos/patología , Neoplasias Encefálicas/enzimología , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patología , Línea Celular Transformada , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/metabolismo , Transformación Celular Neoplásica/patología , Metilación de ADN , Epigénesis Genética , Regulación del Desarrollo de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Técnicas de Silenciamiento del Gen , Humanos , Isocitrato Deshidrogenasa/metabolismo , Factores de Transcripción NFI/genética , Factores de Transcripción NFI/metabolismo , Oligodendroglioma/enzimología , Oligodendroglioma/genética , Oligodendroglioma/patología , Receptor EphA2/genética , Receptor EphA2/metabolismo , Proteínas Represoras/deficienciaRESUMEN
Globally, viral infections substantially contribute to cancer development. Oncogenic viruses are taxonomically heterogeneous and drive cancers using diverse strategies, including epigenomic dysregulation. Here, we discuss how oncogenic viruses disrupt epigenetic homeostasis to drive cancer and focus on how virally mediated dysregulation of host and viral epigenomes impacts the hallmarks of cancer. To illustrate the relationship between epigenetics and viral life cycles, we describe how epigenetic changes facilitate the human papillomavirus (HPV) life cycle and how changes to this process can spur malignancy. We also highlight the clinical impact of virally mediated epigenetic changes on cancer diagnosis, prognosis, and treatment.
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Neoplasias , Virus , Humanos , Virus Oncogénicos/genética , Epigenoma , Neoplasias/patología , Epigénesis Genética , Metilación de ADNRESUMEN
We investigated the role of 3D genome architecture in instructing functional properties of glioblastoma stem cells (GSCs) by generating sub-5-kb resolution 3D genome maps by in situ Hi-C. Contact maps at sub-5-kb resolution allow identification of individual DNA loops, domain organization, and large-scale genome compartmentalization. We observed differences in looping architectures among GSCs from different patients, suggesting that 3D genome architecture is a further layer of inter-patient heterogeneity for glioblastoma. Integration of DNA contact maps with chromatin and transcriptional profiles identified specific mechanisms of gene regulation, including the convergence of multiple super enhancers to individual stemness genes within individual cells. We show that the number of loops contacting a gene correlates with elevated transcription. These results indicate that stemness genes are hubs of interaction between multiple regulatory regions, likely to ensure their sustained expression. Regions of open chromatin common among the GSCs tested were poised for expression of immune-related genes, including CD276 We demonstrate that this gene is co-expressed with stemness genes in GSCs and that CD276 can be targeted with an antibody-drug conjugate to eliminate self-renewing cells. Our results demonstrate that integrated structural genomics data sets can be employed to rationally identify therapeutic vulnerabilities in self-renewing cells.
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Neoplasias Encefálicas/genética , Cromatina/ultraestructura , Mapeo Cromosómico/métodos , Regulación Neoplásica de la Expresión Génica , Glioblastoma/genética , Proteínas de Neoplasias/genética , Antígenos B7/antagonistas & inhibidores , Antígenos B7/genética , Antígenos B7/metabolismo , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patología , Proliferación Celular , Cromatina/química , Elementos de Facilitación Genéticos , Perfilación de la Expresión Génica , Heterogeneidad Genética , Genoma Humano , Genómica/métodos , Glioblastoma/metabolismo , Glioblastoma/patología , Humanos , Terapia Molecular Dirigida , Proteínas de Neoplasias/clasificación , Proteínas de Neoplasias/metabolismo , Células Madre Neoplásicas/metabolismo , Células Madre Neoplásicas/patología , Cultivo Primario de Células , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Transcripción GenéticaRESUMEN
Mantle cell lymphoma (MCL) is an uncommon B-cell non-Hodgkin lymphoma (NHL) that is incurable with standard therapies. The genetic drivers of this cancer have not been firmly established, and the features that contribute to differences in clinical course remain limited. To extend our understanding of the biological pathways involved in this malignancy, we performed a large-scale genomic analysis of MCL using data from 51 exomes and 34 genomes alongside previously published exome cohorts. To confirm our findings, we resequenced the genes identified in the exome cohort in 191 MCL tumors, each having clinical follow-up data. We confirmed the prognostic association of TP53 and NOTCH1 mutations. Our sequencing revealed novel recurrent noncoding mutations surrounding a single exon of the HNRNPH1gene. In RNA-seq data from 103 of these cases, MCL tumors with these mutations had a distinct imbalance of HNRNPH1 isoforms. This altered splicing of HNRNPH1 was associated with inferior outcomes in MCL and showed a significant increase in protein expression by immunohistochemistry. We describe a functional role for these recurrent noncoding mutations in disrupting an autoregulatory feedback mechanism, thereby deregulating HNRNPH1 protein expression. Taken together, these data strongly imply a role for aberrant regulation of messenger RNA processing in MCL pathobiology.
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Predisposición Genética a la Enfermedad/genética , Ribonucleoproteínas Nucleares Heterogéneas/genética , Linfoma de Células del Manto/genética , Adulto , Anciano , Anciano de 80 o más Años , Femenino , Genotipo , Humanos , Masculino , Persona de Mediana Edad , Mutación , Secuenciación Completa del GenomaRESUMEN
The practical application of genome-scale technologies to precision oncology research requires flexible tissue processing strategies that can be used to differentially select both tumour and normal cell populations from formalin-fixed, paraffin-embedded tissues. As tumour sequencing scales towards clinical implementation, practical difficulties in scheduling and obtaining fresh tissue biopsies at scale, including blood samples as surrogates for matched 'normal' DNA, have focused attention on the use of formalin-preserved clinical samples collected routinely for diagnostic purposes. In practice, such samples often contain both tumour and normal cells which, if correctly partitioned, could be used to profile both tumour and normal genomes, thus identifying somatic alterations. Here we report a semi-automated method for laser microdissecting entire slide-mounted tissue sections to enrich for cells of interest with sufficient yield for whole genome and transcriptome sequencing. Using this method, we demonstrated enrichment of tumour material from mixed tumour-normal samples by up to 67%. Leveraging new methods that allow for the extraction of high-quality nucleic acids from small amounts of formalin-fixed tissues, we further showed that the method was successful in yielding sequence data of sufficient quality for use in BC Cancer's Personalized OncoGenomics (POG) program. © 2020 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
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Captura por Microdisección con Láser , Neoplasias/patología , Medicina de Precisión , Animales , Formaldehído , Humanos , Hígado/patología , Ratones , Ratones Endogámicos C57BL , Fijación del TejidoRESUMEN
Glioblastoma multiforme (GBM) is the most deadly brain tumor, and currently lacks effective treatment options. Brain tumor-initiating cells (BTICs) and orthotopic xenografts are widely used in investigating GBM biology and new therapies for this aggressive disease. However, the genomic characteristics and molecular resemblance of these models to GBM tumors remain undetermined. We used massively parallel sequencing technology to decode the genomes and transcriptomes of BTICs and xenografts and their matched tumors in order to delineate the potential impacts of the distinct growth environments. Using data generated from whole-genome sequencing of 201 samples and RNA sequencing of 118 samples, we show that BTICs and xenografts resemble their parental tumor at the genomic level but differ at the mRNA expression and epigenomic levels, likely due to the different growth environment for each sample type. These findings suggest that a comprehensive genomic understanding of in vitro and in vivo GBM model systems is crucial for interpreting data from drug screens, and can help control for biases introduced by cell-culture conditions and the microenvironment in mouse models. We also found that lack of MGMT expression in pretreated GBM is linked to hypermutation, which in turn contributes to increased genomic heterogeneity and requires new strategies for GBM treatment.
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Biomarcadores de Tumor/genética , Neoplasias Encefálicas/patología , Regulación Neoplásica de la Expresión Génica , Genómica/métodos , Glioblastoma/patología , Células Madre Neoplásicas/patología , Microambiente Tumoral/genética , Adulto , Anciano , Anciano de 80 o más Años , Animales , Apoptosis , Neoplasias Encefálicas/genética , Estudios de Casos y Controles , Proliferación Celular , Metilación de ADN , Resistencia a Antineoplásicos , Femenino , Perfilación de la Expresión Génica , Glioblastoma/genética , Humanos , Masculino , Ratones , Ratones SCID , Persona de Mediana Edad , Células Madre Neoplásicas/metabolismo , Transcriptoma , Células Tumorales Cultivadas , Secuenciación Completa del Genoma , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Primary mediastinal large B-cell lymphoma (PMBL) represents a clinically and pathologically distinct subtype of large B-cell lymphomas. Furthermore, molecular studies, including global gene expression profiling, have provided evidence that PMBL is more closely related to classical Hodgkin lymphoma (cHL). Although targeted sequencing studies have revealed a number of mutations involved in PMBL pathogenesis, a comprehensive description of disease-associated genetic alterations and perturbed pathways is still lacking. Here, we performed whole-exome sequencing of 95 PMBL tumors to inform on oncogenic driver genes and recurrent copy number alterations. The integration of somatic gene mutations with gene expression signatures provides further insights into genotype-phenotype interrelation in PMBL. We identified highly recurrent oncogenic mutations in the Janus kinase-signal transducer and activator of transcription and nuclear factor κB pathways, and provide additional evidence of the importance of immune evasion in PMBL (CIITA, CD58, B2M, CD274, and PDCD1LG2). Our analyses highlight the interferon response factor (IRF) pathway as a putative novel hallmark with frequent alterations in multiple pathway members (IRF2BP2, IRF4, and IRF8). In addition, our integrative analysis illustrates the importance of JAK1, RELB, and EP300 mutations driving oncogenic signaling. The identified driver genes were significantly more frequently mutated in PMBL compared with diffuse large B-cell lymphoma, whereas only a limited number of genes were significantly different between PMBL and cHL, emphasizing the close relation between these entities. Our study, performed on a large cohort of PMBL, highlights the importance of distinctive genetic alterations for disease taxonomy with relevance for diagnostic evaluation and therapeutic decision-making.
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Genómica/métodos , Linfoma de Células B Grandes Difuso/genética , Neoplasias del Mediastino/genética , Adolescente , Adulto , Anciano , Estudios de Cohortes , Análisis Mutacional de ADN , Femenino , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Humanos , Linfoma de Células B Grandes Difuso/patología , Masculino , Neoplasias del Mediastino/patología , Persona de Mediana Edad , Mutación , Integración de Sistemas , Adulto JovenRESUMEN
Human cancers, including breast cancers, comprise clones differing in mutation content. Clones evolve dynamically in space and time following principles of Darwinian evolution, underpinning important emergent features such as drug resistance and metastasis. Human breast cancer xenoengraftment is used as a means of capturing and studying tumour biology, and breast tumour xenografts are generally assumed to be reasonable models of the originating tumours. However, the consequences and reproducibility of engraftment and propagation on the genomic clonal architecture of tumours have not been systematically examined at single-cell resolution. Here we show, using deep-genome and single-cell sequencing methods, the clonal dynamics of initial engraftment and subsequent serial propagation of primary and metastatic human breast cancers in immunodeficient mice. In all 15 cases examined, clonal selection on engraftment was observed in both primary and metastatic breast tumours, varying in degree from extreme selective engraftment of minor (<5% of starting population) clones to moderate, polyclonal engraftment. Furthermore, ongoing clonal dynamics during serial passaging is a feature of tumours experiencing modest initial selection. Through single-cell sequencing, we show that major mutation clusters estimated from tumour population sequencing relate predictably to the most abundant clonal genotypes, even in clonally complex and rapidly evolving cases. Finally, we show that similar clonal expansion patterns can emerge in independent grafts of the same starting tumour population, indicating that genomic aberrations can be reproducible determinants of evolutionary trajectories. Our results show that measurement of genomically defined clonal population dynamics will be highly informative for functional studies using patient-derived breast cancer xenoengraftment.
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Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Células Clonales/metabolismo , Células Clonales/patología , Genoma Humano/genética , Análisis de la Célula Individual , Ensayos Antitumor por Modelo de Xenoinjerto , Animales , Neoplasias de la Mama/secundario , Análisis Mutacional de ADN , Genómica , Genotipo , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Ratones , Trasplante de Neoplasias , Factores de Tiempo , Trasplante Heterólogo , Ensayos Antitumor por Modelo de Xenoinjerto/métodosRESUMEN
Tissues used in pathology laboratories are typically stored in the form of formalin-fixed, paraffin-embedded (FFPE) samples. One important consideration in repurposing FFPE material for next generation sequencing (NGS) analysis is the sequencing artifacts that can arise from the significant damage to nucleic acids due to treatment with formalin, storage at room temperature and extraction. One such class of artifacts consists of chimeric reads that appear to be derived from non-contiguous portions of the genome. Here, we show that a major proportion of such chimeric reads align to both the 'Watson' and 'Crick' strands of the reference genome. We refer to these as strand-split artifact reads (SSARs). This study provides a conceptual framework for the mechanistic basis of the genesis of SSARs and other chimeric artifacts along with supporting experimental evidence, which have led to approaches to reduce the levels of such artifacts. We demonstrate that one of these approaches, involving S1 nuclease-mediated removal of single-stranded fragments and overhangs, also reduces sequence bias, base error rates, and false positive detection of copy number and single nucleotide variants. Finally, we describe an analytical approach for quantifying SSARs from NGS data.
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Artefactos , Fijadores , Formaldehído , Secuenciación de Nucleótidos de Alto Rendimiento , Análisis de Secuencia de ADN , Animales , Biblioteca Genómica , Genómica , Calor , Ratones Endogámicos C57BL , Adhesión en ParafinaRESUMEN
BACKGROUND: Aberrations in Capicua (CIC) have recently been implicated as a negative prognostic factor in a multitude of cancer types through the derepression of targets downstream of the mitogen-activated protein kinase (MAPK) signaling cascade, such as oncogenic E26 transformation-specific (ETS) transcription factors. The Ataxin-family protein ATXN1L has previously been reported to interact with CIC in both developmental and disease contexts to facilitate the repression of CIC target genes and promote the post-translational stability of CIC. However, little is known about the mechanisms at the base of ATXN1L-mediated CIC post-translational stability. RESULTS: Functional in vitro studies utilizing ATXN1LKO human cell lines revealed that loss of ATXN1L leads to the accumulation of polyubiquitinated CIC protein, promoting its degradation through the proteasome. Although transcriptomic signatures of ATXN1LKO cell lines indicated upregulation of the mitogen-activated protein kinase pathway, ERK activity was found to contribute to CIC function but not stability. Degradation of CIC protein following loss of ATXN1L was instead observed to be mediated by the E3 ubiquitin ligase TRIM25 which was further validated using glioma-derived cell lines and the TCGA breast carcinoma and liver hepatocellular carcinoma cohorts. CONCLUSIONS: The post-translational regulation of CIC through ATXN1L and TRIM25 independent of ERK activity suggests that the regulation of CIC stability and function is more intricate than previously appreciated and involves several independent pathways. As CIC status has become a prognostic factor in several cancer types, further knowledge into the mechanisms which govern CIC stability and function may prove useful for future therapeutic approaches.
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Sistema de Señalización de MAP Quinasas , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Factores de Transcripción/genética , Proteínas de Motivos Tripartitos/genética , Ubiquitina-Proteína Ligasas/genética , Línea Celular , Humanos , Proteolisis , Factores de Transcripción/metabolismo , Proteínas de Motivos Tripartitos/metabolismo , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
Despite continuing improvements in the management of classical Hodgkin lymphoma (cHL), relapse remains associated with a risk of lymphoma-related mortality. The biological composition of relapse tumour biopsies shows interpatient variability, which can be leveraged to design prognostic biomarkers. Here, we validated the RHL30 assay, a previously reported gene expression model in an independent cohort of 41 patients with relapsed cHL. Patients classified as high-risk by the RHL30 assay had inferior failure-free survival (FFS) after autologous stem cell transplantation (2-year FFS 41% vs. 92%, P = 0·035). The RHL30 model is a robust biomarker that risk-stratifies patients considered for autologous stem cell transplantation.