RESUMO
Ductal carcinoma in situ (DCIS) is a common precursor of invasive breast cancer. Our understanding of its genomic progression to recurrent disease remains poor, partly due to challenges associated with the genomic profiling of formalin-fixed paraffin-embedded (FFPE) materials. Here, we developed Arc-well, a high-throughput single-cell DNA-sequencing method that is compatible with FFPE materials. We validated our method by profiling 40,330 single cells from cell lines, a frozen tissue, and 27 FFPE samples from breast, lung, and prostate tumors stored for 3-31 years. Analysis of 10 patients with matched DCIS and cancers that recurred 2-16 years later show that many primary DCIS had already undergone whole-genome doubling and clonal diversification and that they shared genomic lineages with persistent subclones in the recurrences. Evolutionary analysis suggests that most DCIS cases in our cohort underwent an evolutionary bottleneck, and further identified chromosome aberrations in the persistent subclones that were associated with recurrence.
Assuntos
Neoplasias da Mama , Carcinoma Ductal de Mama , Carcinoma Intraductal não Infiltrante , Feminino , Humanos , Neoplasias da Mama/patologia , Carcinoma Ductal de Mama/genética , Carcinoma Intraductal não Infiltrante/genética , Carcinoma Intraductal não Infiltrante/patologia , Progressão da Doença , Genômica/métodos , Análise da Expressão Gênica de Célula Única , Linhagem Celular TumoralRESUMO
Ductal carcinoma in situ (DCIS) is an early-stage breast cancer that infrequently progresses to invasive ductal carcinoma (IDC). Genomic evolution has been difficult to delineate during invasion due to intratumor heterogeneity and the low number of tumor cells in the ducts. To overcome these challenges, we developed Topographic Single Cell Sequencing (TSCS) to measure genomic copy number profiles of single tumor cells while preserving their spatial context in tissue sections. We applied TSCS to 1,293 single cells from 10 synchronous patients with both DCIS and IDC regions in addition to exome sequencing. Our data reveal a direct genomic lineage between in situ and invasive tumor subpopulations and further show that most mutations and copy number aberrations evolved within the ducts prior to invasion. These results support a multiclonal invasion model, in which one or more clones escape the ducts and migrate into the adjacent tissues to establish the invasive carcinomas.
Assuntos
Neoplasias da Mama/genética , Carcinoma Ductal de Mama/genética , Evolução Clonal , Adulto , Idoso , Neoplasias da Mama/patologia , Carcinoma Ductal de Mama/patologia , Movimento Celular , Exoma , Feminino , Humanos , Pessoa de Meia-Idade , Mutação , Invasividade Neoplásica , Análise de Sequência de DNA , Análise de Célula ÚnicaRESUMO
The adult human breast is comprised of an intricate network of epithelial ducts and lobules that are embedded in connective and adipose tissue1-3. Although most previous studies have focused on the breast epithelial system4-6, many of the non-epithelial cell types remain understudied. Here we constructed the comprehensive Human Breast Cell Atlas (HBCA) at single-cell and spatial resolution. Our single-cell transcriptomics study profiled 714,331 cells from 126 women, and 117,346 nuclei from 20 women, identifying 12 major cell types and 58 biological cell states. These data reveal abundant perivascular, endothelial and immune cell populations, and highly diverse luminal epithelial cell states. Spatial mapping using four different technologies revealed an unexpectedly rich ecosystem of tissue-resident immune cells, as well as distinct molecular differences between ductal and lobular regions. Collectively, these data provide a reference of the adult normal breast tissue for studying mammary biology and diseases such as breast cancer.
Assuntos
Mama , Perfilação da Expressão Gênica , Análise de Célula Única , Adulto , Feminino , Humanos , Mama/citologia , Mama/imunologia , Mama/metabolismo , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Células Endoteliais/classificação , Células Endoteliais/metabolismo , Células Epiteliais/classificação , Células Epiteliais/metabolismo , Genômica , ImunidadeRESUMO
Ductal carcinoma in situ (DCIS) is the most frequently diagnosed early-stage breast cancer. Only a subset of patients progress to invasive ductal carcinoma (IDC), and this presents a formidable clinical challenge for determining which patients to treat aggressively and which patients to monitor without therapeutic intervention. Understanding the molecular and genomic basis of invasion has been difficult to study in DCIS cancers due to several technical obstacles, including low tumour cellularity, lack of fresh-frozen tissues, and intratumour heterogeneity. In this review, we discuss the role of intratumour heterogeneity in the progression of DCIS to IDC in the context of three evolutionary models: independent lineages, evolutionary bottlenecks, and multiclonal invasion. We examine the evidence in support of these models and their relevance to the diagnosis and treatment of patients with DCIS. We also discuss how emerging technologies, such as single-cell sequencing, STAR-FISH, and imaging mass spectrometry, are likely to provide new insights into the evolution of this enigmatic disease. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
Assuntos
Neoplasias da Mama/genética , Carcinoma in Situ/genética , Carcinoma Ductal de Mama/genética , Carcinoma Intraductal não Infiltrante/genética , Genômica , Animais , Neoplasias da Mama/diagnóstico , Neoplasias da Mama/patologia , Carcinoma Ductal de Mama/diagnóstico , Carcinoma Intraductal não Infiltrante/diagnóstico , Progressão da Doença , Feminino , HumanosRESUMO
Background: Principal component analysis (PCA), a standard approach to analysis and visualization of large datasets, is commonly used in biomedical research for detecting similarities and differences among groups of samples. We initially used conventional PCA as a tool for critical quality control of batch and trend effects in multi-omic profiling data produced by The Cancer Genome Atlas (TCGA) project of the NCI. We found, however, that conventional PCA visualizations were often hard to interpret when inter-batch differences were moderate in comparison with intra-batch differences; it was also difficult to quantify batch effects objectively. We, therefore, sought enhancements to make the method more informative in those and analogous settings. Results: We have developed algorithms and a toolbox of enhancements to conventional PCA that improve the detection, diagnosis, and quantitation of differences between or among groups, e.g., groups of molecularly profiled biological samples. The enhancements include (i) computed group centroids; (ii) sample-dispersion rays; (iii) differential coloring of centroids, rays, and sample data points; (iii) trend trajectories; and (iv) a novel separation index (DSC) for quantitation of differences among groups. Conclusions: PCA-Plus has been our most useful single tool for analyzing, visualizing, and quantitating batch effects, trend effects, and class differences in molecular profiling data of many types: mRNA expression, microRNA expression, DNA methylation, and DNA copy number. An early version of PCA-Plus has been used as the central graphical visualization in our MBatch package for near-real-time surveillance of data for analysis working groups in more than 70 TCGA, PanCancer Atlas, PanCancer Analysis of Whole Genomes, and Genome Data Analysis Network projects of the NCI. The algorithms and software are generic, hence applicable more generally to other types of multivariate data as well. PCA-Plus is freely available in a down-loadable R package at our MBatch website.
RESUMO
Obtaining a parts list of the sensory components of the retina is vital to understand the effects of light in behavior, health, and disease. Rods, cones, and intrinsically photosensitive retinal ganglion cells (ipRGCs) are the best described photoreceptors in the mammalian retina, but recent functional roles have been proposed for retinal neuropsin (Opn5)-an atypical opsin. However, little is known about the pattern of Opn5 expression in the retina. Using cre (Opn5cre ) and cre-dependent reporters, we uncover patterns of Opn5 expression and find that Opn5 is restricted to retinal ganglion cells (RGCs). Opn5-RGCs are nonhomogenously distributed through the retina, with greater densities of cells located in the dorsotemporal quadrant. In addition to the local topology of these cells, using cre-dependent AAV viral tracing, we surveyed their central targets and found that they are biased towards image-forming and image-stabilizing regions. Finally, molecular and electrophysiological profiling reveal that Opn5-RGCs comprise previously defined RGC types that respond optimally to edges and object-motion (F-mini-ONs, HD2, HD1, LEDs, ooDSRGCs, etc.). Together, these data describe the second collection of RGCs that express atypical opsins in the mouse, and expand the roles of image-forming cells in retinal physiology and function.
Assuntos
Retina , Células Ganglionares da Retina , Animais , Mamíferos , Proteínas de Membrana/metabolismo , Camundongos , Opsinas/genética , Opsinas/metabolismo , Retina/fisiologia , Células Fotorreceptoras Retinianas Cones/metabolismo , Células Ganglionares da Retina/fisiologia , Opsinas de Bastonetes/metabolismoRESUMO
Ductal carcinoma in situ (DCIS) is a non-invasive breast neoplasia that accounts for 25% of all screen-detected breast cancers diagnosed annually. Neoplastic cells in DCIS are confined to the ductal system of the breast, although they can escape and progress to invasive breast cancer in a subset of patients. A key concern of DCIS is overtreatment, as most patients screened for DCIS and in whom DCIS is diagnosed will not go on to exhibit symptoms or die of breast cancer, even if left untreated. However, differentiating low-risk, indolent DCIS from potentially progressive DCIS remains challenging. In this Review, we summarize our current knowledge of DCIS and explore open questions about the basic biology of DCIS, including those regarding how genomic events in neoplastic cells and the surrounding microenvironment contribute to the progression of DCIS to invasive breast cancer. Further, we discuss what information will be needed to prevent overtreatment of indolent DCIS lesions without compromising adequate treatment for high-risk patients.
Assuntos
Neoplasias da Mama , Carcinoma Intraductal não Infiltrante , Humanos , Feminino , Carcinoma Intraductal não Infiltrante/diagnóstico , Carcinoma Intraductal não Infiltrante/genética , Carcinoma Intraductal não Infiltrante/patologia , Neoplasias da Mama/diagnóstico , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Microambiente TumoralRESUMO
Ductal carcinoma in situ (DCIS) is the most common form of preinvasive breast cancer and, despite treatment, a small fraction (5-10%) of DCIS patients develop subsequent invasive disease. A fundamental biologic question is whether the invasive disease arises from tumor cells in the initial DCIS or represents new unrelated disease. To address this question, we performed genomic analyses on the initial DCIS lesion and paired invasive recurrent tumors in 95 patients together with single-cell DNA sequencing in a subset of cases. Our data show that in 75% of cases the invasive recurrence was clonally related to the initial DCIS, suggesting that tumor cells were not eliminated during the initial treatment. Surprisingly, however, 18% were clonally unrelated to the DCIS, representing new independent lineages and 7% of cases were ambiguous. This knowledge is essential for accurate risk evaluation of DCIS, treatment de-escalation strategies and the identification of predictive biomarkers.
Assuntos
Neoplasias da Mama , Carcinoma Ductal de Mama , Carcinoma Intraductal não Infiltrante , Biomarcadores Tumorais/análise , Biomarcadores Tumorais/genética , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Carcinoma Ductal de Mama/genética , Carcinoma Intraductal não Infiltrante/genética , Carcinoma Intraductal não Infiltrante/patologia , Feminino , Genômica , Humanos , Recidiva Local de Neoplasia/genéticaRESUMO
In several areas of the central nervous system, neurons are regionally organized into groups or layers that carry out specific activities. In this form of patterning, neurons of distinct types localize their cell bodies to just one or a few of the layers within a structure. However, little is known about whether diverse neuron types within a lamina share molecular features that coordinate their organization. To begin to identify such candidates, we used the laminated murine retina to screen 92 lacZ reporter lines available through the Knockout Mouse Project. Thirty-two of these displayed reporter expression in restricted subsets of inner retina neurons. We then identified the spatiotemporal expression patterns of these genes at key developmental stages. This uncovered several that were heavily enriched in development but reduced in adulthood, including the transcriptional regulator Hmga1. An additional set of genes displayed maturation associated laminar enrichment. Among these, we identified Bbox1 as a novel gene that specifically labels all neurons in the ganglion cell layer but is largely excluded from otherwise molecularly similar neurons in the inner retina. Finally, we established Dbn1 as a new marker enriched in amacrines and Fmnl3 as a marker for subsets of αRGCs. Together, these data provide a spatiotemporal map for laminae-specific molecules and suggest that diverse neuron types within a lamina share coordinating molecular features that may inform their fate or function.
Assuntos
Neurônios Retinianos/citologia , Animais , CamundongosRESUMO
Structural changes in pre and postsynaptic neurons that accompany synapse formation often temporally and spatially overlap. Thus, it has been difficult to resolve which processes drive patterned connectivity. To overcome this, we use the laminated outer murine retina. We identify the serine/threonine kinase LKB1 as a key driver of synapse layer emergence. The absence of LKB1 in the retina caused a marked mislocalization and delay in synapse layer formation. In parallel, LKB1 modulated postsynaptic horizontal cell refinement and presynaptic photoreceptor axon growth. Mislocalized horizontal cell processes contacted aberrant cone axons in LKB1 mutants. These defects coincided with altered synapse protein organization, and horizontal cell neurites were misdirected to ectopic synapse protein regions. Together, these data suggest that LKB1 instructs the timing and location of connectivity in the outer retina via coordinate regulation of pre and postsynaptic neuron structure and the localization of synapse-associated proteins.