RESUMO
Tandem repeats of simple sequence motifs, also known as microsatellites, are abundant in the genome. Because their repeat structure makes replication error-prone, variant microsatellite lengths are often generated during germline and other somatic expansions. As such, microsatellite length variations can serve as markers for cancer. However, accurate error-free measurement of microsatellite lengths is difficult with current methods precisely because of this high error rate during amplification. We have solved this problem by using partial mutagenesis to disrupt enough of the repeat structure of initial templates so that their sequence lengths replicate faithfully. In this work, we use bisulfite mutagenesis to convert a C to a U, later read as T. Compared to untreated templates, we achieve three orders of magnitude reduction in the error rate per round of replication. By requiring agreement from two independent first copies of an initial template, we reach error rates below one in a million. We apply this method to a thousand microsatellite loci from the human genome, revealing microsatellite length distributions not observable without mutagenesis.
Assuntos
Genoma Humano , Repetições de Microssatélites , Mutagênese Sítio-Dirigida , Humanos , Repetições de Microssatélites/genética , Mutagênese Sítio-Dirigida/métodosRESUMO
Pancreatic ductal adenocarcinoma (PDAC) is the most lethal common malignancy, with little improvement in patient outcomes over the past decades. Recently, subtypes of pancreatic cancer with different prognoses have been elaborated; however, the inability to model these subtypes has precluded mechanistic investigation of their origins. Here, we present a xenotransplantation model of PDAC in which neoplasms originate from patient-derived organoids injected directly into murine pancreatic ducts. Our model enables distinction of the two main PDAC subtypes: intraepithelial neoplasms from this model progress in an indolent or invasive manner representing the classical or basal-like subtypes of PDAC, respectively. Parameters that influence PDAC subtype specification in this intraductal model include cell plasticity and hyperactivation of the RAS pathway. Finally, through intratumoral dissection and the direct manipulation of RAS gene dosage, we identify a suite of RAS-regulated secreted and membrane-bound proteins that may represent potential candidates for therapeutic intervention in patients with PDAC. SIGNIFICANCE: Accurate modeling of the molecular subtypes of pancreatic cancer is crucial to facilitate the generation of effective therapies. We report the development of an intraductal organoid transplantation model of pancreatic cancer that models the progressive switching of subtypes, and identify stochastic and RAS-driven mechanisms that determine subtype specification.See related commentary by Pickering and Morton, p. 1448.This article is highlighted in the In This Issue feature, p. 1426.
Assuntos
Adenocarcinoma/genética , Regulação Neoplásica da Expressão Gênica/genética , Ductos Pancreáticos/transplante , Animais , Carcinoma Ductal Pancreático , Modelos Animais de Doenças , Humanos , Camundongos , PrognósticoRESUMO
PURPOSE: Copy-number profiling of multiple individual cells from sparse sequencing may be used to reveal a detailed picture of genomic heterogeneity and clonal organization in a tissue biopsy specimen. We sought to provide a comprehensive computational pipeline for single-cell genomics, to facilitate adoption of this molecular technology for basic and translational research. MATERIALS AND METHODS: The pipeline comprises software tools programmed in Python and in R and depends on Bowtie, HISAT2, Matplotlib, and Qt. It is installed and used with Anaconda. RESULTS: Here we describe a complete pipeline for sparse single-cell genomic data, encompassing all steps of single-nucleus DNA copy-number profiling, from raw sequence processing to clonal structure analysis and visualization. For the latter, a specialized graphical user interface termed the single-cell genome viewer (SCGV) is provided. With applications to cancer diagnostics in mind, the SCGV allows for zooming and linkage to the University of California at Santa Cruz Genome Browser from each of the multiple integrated views of single-cell copy-number profiles. The latter can be organized by clonal substructure or by any of the associated metadata such as anatomic location and histologic characterization. CONCLUSION: The pipeline is available as open-source software for Linux and OS X. Its modular structure, extensive documentation, and ease of deployment using Anaconda facilitate its adoption by researchers and practitioners of single-cell genomics. With open-source availability and Massachusetts Institute of Technology licensing, it provides a basis for additional development by the cancer bioinformatics community.
Assuntos
Biologia Computacional , Software , Genoma , Genômica , HumanosRESUMO
Copy number alterations (CNAs) play an important role in molding the genomes of breast cancers and have been shown to be clinically useful for prognostic and therapeutic purposes. However, our knowledge of intra-tumoral genetic heterogeneity of this important class of somatic alterations is limited. Here, using single-cell sequencing, we comprehensively map out the facets of copy number alteration heterogeneity in a cohort of breast cancer tumors. Ou/var/www/html/elife/12-05-2020/backup/r analyses reveal: genetic heterogeneity of non-tumor cells (i.e. stroma) within the tumor mass; the extent to which copy number heterogeneity impacts breast cancer genomes and the importance of both the genomic location and dosage of sub-clonal events; the pervasive nature of genetic heterogeneity of chromosomal amplifications; and the association of copy number heterogeneity with clinical and biological parameters such as polyploidy and estrogen receptor negative status. Our data highlight the power of single-cell genomics in dissecting, in its many forms, intra-tumoral genetic heterogeneity of CNAs, the magnitude with which CNA heterogeneity affects the genomes of breast cancers, and the potential importance of CNA heterogeneity in phenomena such as therapeutic resistance and disease relapse.
Cells in the body remain healthy by tightly preventing and repairing random changes, or mutations, in their genetic material. In cancer cells, however, these mechanisms can break down. When these cells grow and multiply, they can then go on to accumulate many mutations. As a result, cancer cells in the same tumor can each contain a unique combination of genetic changes. This genetic heterogeneity has the potential to affect how cancer responds to treatment, and is increasingly becoming appreciated clinically. For example, if a drug only works against cancer cells carrying a specific mutation, any cells lacking this genetic change will keep growing and cause a relapse. However, it is still difficult to quantify and understand genetic heterogeneity in cancer. Copy number alterations (or CNAs) are a class of mutation where large and small sections of genetic material are gained or lost. This can result in cells that have an abnormal number of copies of the genes in these sections. Here, Baslan et al. set out to explore how CNAs might vary between individual cancer cells within the same tumor. To do so, thousands of individual cancer cells were isolated from human breast tumors, and a technique called single-cell genome sequencing used to screen the genetic information of each of them. These experiments confirmed that CNAs did differ sometimes dramatically between patients and among cells taken from the same tumor. For example, many of the cells carried extra copies of well-known cancer genes important for treatment, but the exact number of copies varied between cells. This heterogeneity existed for individual genes as well as larger stretches of DNA: this was the case, for instance, for an entire section of chromosome 8, a region often affected in breast and other tumors. The work by Baslan et al. captures the sheer extent of genetic heterogeneity in cancer and in doing so, highlights the power of single-cell genome sequencing. In the future, a finer understanding of the genetic changes present at the level of an individual cancer cell may help clinicians to manage the disease more effectively.
Assuntos
Biomarcadores Tumorais/genética , Neoplasias da Mama/genética , Variações do Número de Cópias de DNA , Dosagem de Genes , Heterogeneidade Genética , Genômica , Análise de Célula Única , Sequenciamento Completo do Genoma , Neoplasias da Mama/mortalidade , Neoplasias da Mama/patologia , Neoplasias da Mama/terapia , Ensaios Clínicos Fase II como Assunto , Feminino , Predisposição Genética para Doença , Humanos , Fenótipo , Prognóstico , RNA-SeqRESUMO
Measuring minimal residual disease in cancer has applications for prognosis, monitoring treatment and detection of recurrence. Simple sequence-based methods to detect nucleotide substitution variants have error rates (about 10-3) that limit sensitive detection. We developed and characterized the performance of MASQ (multiplex accurate sensitive quantitation), a method with an error rate below 10-6. MASQ counts variant templates accurately in the presence of millions of host genomes by using tags to identify each template and demanding consensus over multiple reads. Since the MASQ protocol multiplexes 50 target loci, we can both integrate signal from multiple variants and capture subclonal response to treatment. Compared to existing methods for variant detection, MASQ achieves an excellent combination of sensitivity, specificity and yield. We tested MASQ in a pilot study in acute myeloid leukemia (AML) patients who entered complete remission. We detect leukemic variants in the blood and bone marrow samples of all five patients, after induction therapy, at levels ranging from 10-2 to nearly 10-6. We observe evidence of sub-clonal structure and find higher target variant frequencies in patients who go on to relapse, demonstrating the potential for MASQ to quantify residual disease in AML.
Assuntos
Leucemia Mieloide Aguda/genética , Algoritmos , Genômica/métodos , Humanos , Leucemia Mieloide Aguda/terapia , Mutação , Neoplasia Residual , Projetos Piloto , Recidiva , Indução de Remissão , Sequenciamento Completo do GenomaRESUMO
High levels of cancer aneuploidy are frequently associated with poor prognosis. To examine the relationship between aneuploidy and cancer progression, we analyzed a series of congenic cell lines that harbor single extra chromosomes. We found that across 13 different trisomic cell lines, 12 trisomies suppressed invasiveness or were largely neutral, while a single trisomy increased metastatic behavior by triggering a partial epithelial-mesenchymal transition. In contrast, we discovered that chromosomal instability activates cGAS/STING signaling but strongly suppresses invasiveness. By analyzing patient copy-number data, we demonstrate that specific aneuploidies are associated with distinct outcomes, and the acquisition of certain aneuploidies is in fact linked with a favorable prognosis. Thus, aneuploidy is not a uniform driver of malignancy, and different aneuploidies can uniquely influence tumor progression. At the same time, the gain of a single chromosome is capable of inducing a profound cell state transition, thereby linking genomic plasticity, phenotypic plasticity, and metastasis.
Assuntos
Aneuploidia , Movimento Celular , Instabilidade Cromossômica , Cromossomos Humanos Par 5/genética , Neoplasias do Colo/patologia , Neoplasias do Colo/prevenção & controle , Transição Epitelial-Mesenquimal , Animais , Apoptose , Proliferação de Células , Neoplasias do Colo/genética , Feminino , Humanos , Masculino , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Nus , Invasividade Neoplásica , Nucleotidiltransferases/genética , Nucleotidiltransferases/metabolismo , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
We show the use of 5'-Acrydite oligonucleotides to copolymerize single-cell DNA or RNA into balls of acrylamide gel (BAGs). Combining this step with split-and-pool techniques for creating barcodes yields a method with advantages in cost and scalability, depth of coverage, ease of operation, minimal cross-contamination, and efficient use of samples. We perform DNA copy number profiling on mixtures of cell lines, nuclei from frozen prostate tumors, and biopsy washes. As applied to RNA, the method has high capture efficiency of transcripts and sufficient consistency to clearly distinguish the expression patterns of cell lines and individual nuclei from neurons dissected from the mouse brain. By using varietal tags (UMIs) to achieve sequence error correction, we show extremely low levels of cross-contamination by tracking source-specific SNVs. The method is readily modifiable, and we will discuss its adaptability and diverse applications.
Assuntos
Acrilamida , Ácidos Nucleicos , Análise de Célula Única/métodos , Acrilamida/química , DNA , Contaminação por DNA , Variações do Número de Cópias de DNA , Dosagem de Genes , Perfilação da Expressão Gênica/métodos , Perfilação da Expressão Gênica/normas , Biblioteca Gênica , Humanos , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patologia , Ácidos Nucleicos/química , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Análise de Sequência com Séries de Oligonucleotídeos/normas , Polimerização , RNA , Análise de Célula Única/normasRESUMO
A distinction between indolent and aggressive disease is a major challenge in diagnostics of prostate cancer. As genetic heterogeneity and complexity may influence clinical outcome, we have initiated studies on single tumor cell genomics. In this study, we demonstrate that sparse DNA sequencing of single-cell nuclei from prostate core biopsies is a rich source of quantitative parameters for evaluating neoplastic growth and aggressiveness. These include the presence of clonal populations, the phylogenetic structure of those populations, the degree of the complexity of copy-number changes in those populations, and measures of the proportion of cells with clonal copy-number signatures. The parameters all showed good correlation to the measure of prostatic malignancy, the Gleason score, derived from individual prostate biopsy tissue cores. Remarkably, a more accurate histopathologic measure of malignancy, the surgical Gleason score, agrees better with these genomic parameters of diagnostic biopsy than it does with the diagnostic Gleason score and related measures of diagnostic histopathology. This is highly relevant because primary treatment decisions are dependent upon the biopsy and not the surgical specimen. Thus, single-cell analysis has the potential to augment traditional core histopathology, improving both the objectivity and accuracy of risk assessment and inform treatment decisions.Significance: Genomic analysis of multiple individual cells harvested from prostate biopsies provides an indepth view of cell populations comprising a prostate neoplasm, yielding novel genomic measures with the potential to improve the accuracy of diagnosis and prognosis in prostate cancer. Cancer Res; 78(2); 348-58. ©2017 AACR.
Assuntos
Biomarcadores Tumorais/genética , Genômica/métodos , Neoplasias da Próstata/diagnóstico , Análise de Célula Única/métodos , Idoso , Idoso de 80 Anos ou mais , Humanos , Masculino , Pessoa de Meia-Idade , Gradação de Tumores , Estadiamento de Neoplasias , Filogenia , Prostatectomia , Neoplasias da Próstata/genética , Neoplasias da Próstata/cirurgia , Medição de RiscoRESUMO
Here, we explore the potential of single-cell genomic analysis in blood for early detection of cancer; we consider a method that screens the presence of recurrent patterns of copy number (CN) alterations using sparse single-cell sequencing. We argue for feasibility, based on in silico analysis of existing single-cell data and cancer CN profiles. Sampling procedures from existing diploid single cells can render data for a cell with any given profile. Sampling from multiple published tumor profiles can interrogate cancer clonality via an algorithm that tests the multiplicity of close pairwise similarities among single-cell cancer genomes. The majority of common solid cancers would be detectable in this manner. As any early detection method must be verifiable and actionable, we describe how further analysis of suspect cells can aid in determining risk and anatomic origin. Future affordability rests on currently available procedures for tumor cell enrichment and inexpensive methods for single-cell analysis.
Assuntos
Simulação por Computador , Dosagem de Genes , Genoma Humano , Neoplasias , Animais , Humanos , Neoplasias/diagnóstico , Neoplasias/genéticaRESUMO
Copy number variants (CNVs) underlie a significant amount of genetic diversity and disease. CNVs can be detected by a number of means, including chromosomal microarray analysis (CMA) and whole-genome sequencing (WGS), but these approaches suffer from either limited resolution (CMA) or are highly expensive for routine screening (both CMA and WGS). As an alternative, we have developed a next-generation sequencing-based method for CNV analysis termed SMASH, for short multiply aggregated sequence homologies. SMASH utilizes random fragmentation of input genomic DNA to create chimeric sequence reads, from which multiple mappable tags can be parsed using maximal almost-unique matches (MAMs). The SMASH tags are then binned and segmented, generating a profile of genomic copy number at the desired resolution. Because fewer reads are necessary relative to WGS to give accurate CNV data, SMASH libraries can be highly multiplexed, allowing large numbers of individuals to be analyzed at low cost. Increased genomic resolution can be achieved by sequencing to higher depth.
Assuntos
Dosagem de Genes , Análise de Sequência de DNA , Linhagem Celular Tumoral , Biologia Computacional , Variações do Número de Cópias de DNA , Feminino , Genoma Humano , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Masculino , SoftwareRESUMO
We present Ginkgo (http://qb.cshl.edu/ginkgo), a user-friendly, open-source web platform for the analysis of single-cell copy-number variations (CNVs). Ginkgo automatically constructs copy-number profiles of cells from mapped reads and constructs phylogenetic trees of related cells. We validated Ginkgo by reproducing the results of five major studies. After comparing three commonly used single-cell amplification techniques, we concluded that degenerate oligonucleotide-primed PCR is the most consistent for CNV analysis.
Assuntos
Biologia Computacional , Variações do Número de Cópias de DNA , Genoma Humano , Oligonucleotídeos/genética , Algoritmos , Animais , Automação , Análise por Conglomerados , Drosophila , Feminino , Dosagem de Genes , Genoma , Humanos , Internet , Neoplasias Pulmonares/diagnóstico , Neoplasias Pulmonares/genética , Masculino , Camundongos , Pan troglodytes , Filogenia , Reação em Cadeia da Polimerase , Ratos , Reprodutibilidade dos Testes , Cromossomos Sexuais , Carcinoma de Pequenas Células do Pulmão/diagnóstico , Carcinoma de Pequenas Células do Pulmão/genética , SoftwareRESUMO
Genome-wide analysis at the level of single cells has recently emerged as a powerful tool to dissect genome heterogeneity in cancer, neurobiology, and development. To be truly transformative, single-cell approaches must affordably accommodate large numbers of single cells. This is feasible in the case of copy number variation (CNV), because CNV determination requires only sparse sequence coverage. We have used a combination of bioinformatic and molecular approaches to optimize single-cell DNA amplification and library preparation for highly multiplexed sequencing, yielding a method that can produce genome-wide CNV profiles of up to a hundred individual cells on a single lane of an Illumina HiSeq instrument. We apply the method to human cancer cell lines and biopsied cancer tissue, thereby illustrating its efficiency, reproducibility, and power to reveal underlying genetic heterogeneity and clonal phylogeny. The capacity of the method to facilitate the rapid profiling of hundreds to thousands of single-cell genomes represents a key step in making single-cell profiling an easily accessible tool for studying cell lineage.
Assuntos
Variações do Número de Cópias de DNA , DNA de Neoplasias/genética , Reação em Cadeia da Polimerase Multiplex/métodos , Análise de Sequência de DNA/métodos , Análise de Célula Única/métodos , Algoritmos , Sequência de Bases , Linhagem Celular Tumoral , Genoma Humano , Humanos , Dados de Sequência MolecularRESUMO
In situ detection of genomic alterations in cancer provides information at the single cell level, making it possible to investigate genomic changes in cells in a tissue context. Such topological information is important when studying intratumor heterogeneity as well as alterations related to different steps in tumor progression. We developed a quantitative multigene fluorescence in situ hybridization (QM FISH) method to detect multiple genomic regions in single cells in complex tissues. As a "proof of principle" we applied the method to breast cancer samples to identify partners in whole arm (WA) translocations. WA gain of chromosome arm 1q and loss of chromosome arm 16q are among the most frequent genomic events in breast cancer. By designing five specific FISH probes based on breakpoint information from comparative genomic hybridization array (aCGH) profiles, we visualized chromosomal translocations in clinical samples at the single cell level. By analyzing aCGH data from 295 patients with breast carcinoma with known molecular subtype, we found concurrent WA gain of 1q and loss of 16q to be more frequent in luminal A tumors compared to other molecular subtypes. QM FISH applied to a subset of samples (n = 26) identified a derivative chromosome der(1;16)(q10;p10), a result of a centromere-close translocation between chromosome arms 1q and 16p. In addition, we observed that the distribution of cells with the translocation varied from sample to sample, some had a homogenous cell population while others displayed intratumor heterogeneity with cell-to-cell variation. Finally, for one tumor with both preinvasive and invasive components, the fraction of cells with translocation was lower and more heterogeneous in the preinvasive tumor cells compared to the cells in the invasive component.
Assuntos
Neoplasias da Mama/genética , Hibridização in Situ Fluorescente/métodos , Translocação Genética , Quebra Cromossômica , Cromossomos Humanos Par 1 , Cromossomos Humanos Par 16 , Feminino , HumanosRESUMO
Timely characterization of a cancer's evolution is required to predict treatment efficacy and to detect resistance early. High content analysis of single Circulating Tumor Cells (CTCs) enables sequential characterization of genotypic, morphometric and protein expression alterations in real time over the course of cancer treatment. This concept was investigated in a patient with castrate-resistant prostate cancer progressing through both chemotherapy and targeted therapy. In this case study, we integrate across four timepoints 41 genome-wide copy number variation (CNV) profiles plus morphometric parameters and androgen receptor (AR) protein levels. Remarkably, little change was observed in response to standard chemotherapy, evidenced by the fact that a unique clone (A), exhibiting highly rearranged CNV profiles and AR+ phenotype was found circulating before and after treatment. However, clinical response and subsequent progression after targeted therapy was associated with the drastic depletion of clone A, followed by the sequential emergence of two distinct CTC sub-populations that differed in both AR genotype and expression phenotype. While AR- cells with flat or pseudo-diploid CNV profiles (clone B) were identified at the time of response, a new tumor lineage of AR+ cells (clone C) with CNV altered profiles was detected during relapse. We showed that clone C, despite phylogenetically related to clone A, possessed a unique set of somatic CNV alterations, including MYC amplification, an event linked to hormone escape. Interesting, we showed that both clones acquired AR gene amplification by deploying different evolutionary paths. Overall, these data demonstrate the timeframe of tumor evolution in response to therapy and provide a framework for the multi-scale analysis of fluid biopsies to quantify and monitor disease evolution in individual patients.
Assuntos
Genômica , Células Neoplásicas Circulantes/metabolismo , Fenótipo , Neoplasias da Próstata/diagnóstico , Neoplasias da Próstata/genética , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Aberrações Cromossômicas , Evolução Clonal , Variações do Número de Cópias de DNA , Humanos , Imuno-Histoquímica , Espaço Intracelular , Masculino , Metástase Neoplásica , Células Neoplásicas Circulantes/patologia , Neoplasias da Próstata/terapia , Neoplasias de Próstata Resistentes à Castração , Transporte Proteico , Receptores Androgênicos/metabolismo , Análise de Célula ÚnicaRESUMO
Cancer cells frequently depend on chromatin regulatory activities to maintain a malignant phenotype. Here, we show that leukemia cells require the mammalian SWI/SNF chromatin remodeling complex for their survival and aberrant self-renewal potential. While Brg1, an ATPase subunit of SWI/SNF, is known to suppress tumor formation in several cell types, we found that leukemia cells instead rely on Brg1 to support their oncogenic transcriptional program, which includes Myc as one of its key targets. To account for this context-specific function, we identify a cluster of lineage-specific enhancers located 1.7 Mb downstream from Myc that are occupied by SWI/SNF as well as the BET protein Brd4. Brg1 is required at these distal elements to maintain transcription factor occupancy and for long-range chromatin looping interactions with the Myc promoter. Notably, these distal Myc enhancers coincide with a region that is focally amplified in â¼3% of acute myeloid leukemias. Together, these findings define a leukemia maintenance function for SWI/SNF that is linked to enhancer-mediated gene regulation, providing general insights into how cancer cells exploit transcriptional coactivators to maintain oncogenic gene expression programs.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Elementos Facilitadores Genéticos/fisiologia , Regulação Neoplásica da Expressão Gênica , Leucemia Mieloide Aguda/fisiopatologia , Proteínas Proto-Oncogênicas c-myc/genética , Fatores de Transcrição/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , DNA Helicases/genética , DNA Helicases/metabolismo , Proteínas de Ligação a DNA/genética , Elementos Facilitadores Genéticos/genética , Técnicas de Silenciamento de Genes , Humanos , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Regiões Promotoras Genéticas/genética , Ligação Proteica , Proteínas Proto-Oncogênicas c-myc/metabolismo , Fatores de Transcrição/genéticaRESUMO
Finding regions of the genome that are significantly recurrent in noisy data are a common but difficult problem in present day computational biology. Cores of recurrent events (CORE) is a computational approach to solving this problem that is based on a formalized notion by which "core" intervals explain the observed data, where the number of cores is the "depth" of the explanation. Given that formalization, we implement CORE as a combinatorial optimization procedure with depth chosen from considerations of statistical significance. An important feature of CORE is its ability to explain data with cores of widely varying lengths. We examine the performance of this system with synthetic data, and then provide two demonstrations of its utility with actual data. Applying CORE to a collection of DNA copy number profiles from single cells of a given tumor, we determine tumor population phylogeny and find the features that separate subpopulations. Applying CORE to comparative genomic hybridization data from a large set of tumor samples, we define regions of recurrent copy number aberration in breast cancer.
Assuntos
Neoplasias da Mama/genética , Regulação Neoplásica da Expressão Gênica , Genômica/métodos , Modelos Genéticos , Neoplasias da Mama/secundário , Hibridização Genômica Comparativa/métodos , Biologia Computacional/métodos , Variações do Número de Cópias de DNA/genética , Bases de Dados Genéticas , Feminino , Humanos , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Filogenia , Software , TranscriptomaAssuntos
Diferenciação Celular/fisiologia , Transformação Celular Neoplásica/patologia , Células-Tronco Neoplásicas/patologia , Células-Tronco Neoplásicas/fisiologia , Animais , Ciclo Celular/fisiologia , Regulação Neoplásica da Expressão Gênica/fisiologia , Humanos , Metástase Neoplásica/fisiopatologiaRESUMO
Although heritable factors are an important determinant of risk of early-onset cancer, the majority of these malignancies appear to occur sporadically without identifiable risk factors. Germline de novo copy-number variations (CNVs) have been observed in sporadic neurocognitive and cardiovascular disorders. We explored this mechanism in 382 genomes of 116 early-onset cancer case-parent trios and unaffected siblings. Unique de novo germline CNVs were not observed in 107 breast or colon cancer trios or controls but were indeed found in 7% of 43 testicular germ cell tumor trios; this percentage exceeds background CNV rates and suggests a rare de novo genetic paradigm for susceptibility to some human malignancies.
Assuntos
Variações do Número de Cópias de DNA/genética , Predisposição Genética para Doença/genética , Genômica/métodos , Mutação em Linhagem Germinativa/genética , Neoplasias Testiculares/genética , Adulto , Humanos , Masculino , Pais , Projetos de PesquisaRESUMO
The large chromosomal deletions frequently observed in cancer genomes are often thought to arise as a "two-hit" mechanism in the process of tumor-suppressor gene (TSG) inactivation. Using a murine model system of hepatocellular carcinoma (HCC) and in vivo RNAi, we test an alternative hypothesis, that such deletions can arise from selective pressure to attenuate the activity of multiple genes. By targeting the mouse orthologs of genes frequently deleted on human 8p22 and adjacent regions, which are lost in approximately half of several other major epithelial cancers, we provide evidence suggesting that multiple genes on chromosome 8p can cooperatively inhibit tumorigenesis in mice, and that their cosuppression can synergistically promote tumor growth. In addition, in human HCC patients, the combined down-regulation of functionally validated 8p TSGs is associated with poor survival, in contrast to the down-regulation of any individual gene. Our data imply that large cancer-associated deletions can produce phenotypes distinct from those arising through loss of a single TSG, and as such should be considered and studied as distinct mutational events.
Assuntos
Carcinoma Hepatocelular/genética , Deleção de Genes , Genes Supressores de Tumor/fisiologia , Genômica/métodos , Neoplasias Hepáticas Experimentais/genética , Monossomia , Animais , Carcinoma Hepatocelular/mortalidade , Linhagem Celular Transformada , Linhagem Celular Tumoral , Cromossomos Humanos Par 8 , Feminino , Regulação Neoplásica da Expressão Gênica/genética , Haploinsuficiência/genética , Humanos , Fígado/citologia , Neoplasias Hepáticas Experimentais/mortalidade , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Nus , Interferência de RNA , Células-Tronco/citologiaRESUMO
Genomic analysis provides insights into the role of copy number variation in disease, but most methods are not designed to resolve mixed populations of cells. In tumours, where genetic heterogeneity is common, very important information may be lost that would be useful for reconstructing evolutionary history. Here we show that with flow-sorted nuclei, whole genome amplification and next generation sequencing we can accurately quantify genomic copy number within an individual nucleus. We apply single-nucleus sequencing to investigate tumour population structure and evolution in two human breast cancer cases. Analysis of 100 single cells from a polygenomic tumour revealed three distinct clonal subpopulations that probably represent sequential clonal expansions. Additional analysis of 100 single cells from a monogenomic primary tumour and its liver metastasis indicated that a single clonal expansion formed the primary tumour and seeded the metastasis. In both primary tumours, we also identified an unexpectedly abundant subpopulation of genetically diverse 'pseudodiploid' cells that do not travel to the metastatic site. In contrast to gradual models of tumour progression, our data indicate that tumours grow by punctuated clonal expansions with few persistent intermediates.