RESUMO
Super-enhancers (SEs) are expansive regions of genomic DNA that regulate the expression of genes involved in cell identity and cell fate. We recently identified developmental stage- and cell type-specific modules within the murine Vsx2 SE. Here, we show that the human VSX2 SE modules have similar developmental stage- and cell type-specific activity in reporter gene assays. By inserting the human sequence of one VSX2 SE module into a mouse with microphthalmia, eye size was rescued. To understand the function of these SE modules during human retinal development, we deleted individual modules in human embryonic stem cells and generated retinal organoids. Deleting one module results in small organoids, recapitulating the small-eyed phenotype of mice with microphthalmia, while deletion of the other module led to disruptions in bipolar neuron development. This prototypical SE serves as a model for understanding developmental stage- and cell type-specific effects of neurogenic transcription factors with complex expression patterns. Moreover, by elucidating the gene regulatory mechanisms, we can begin to examine how dysregulation of these mechanisms contributes to phenotypic diversity and disease.
Assuntos
Elementos Facilitadores Genéticos , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio , Retina , Fatores de Transcrição , Animais , Humanos , Camundongos , Elementos Facilitadores Genéticos/genética , Evolução Molecular , Proteínas de Homeodomínio/metabolismo , Proteínas de Homeodomínio/genética , Células-Tronco Embrionárias Humanas/metabolismo , Células-Tronco Embrionárias Humanas/citologia , Microftalmia/genética , Microftalmia/patologia , Neurogênese/genética , Organoides/metabolismo , Retina/metabolismo , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genéticaRESUMO
Most patients with follicular lymphoma (FL) experience multiple relapses necessitating subsequent lines of therapy. Ibrutinib, a Bruton tyrosine kinase (BTK) inhibitor approved for the treatment of several B-cell malignancies, showed promising activity in FL in a phase 1 study. We report the results of a phase 2 trial evaluating ibrutinib in recurrent FL. Forty patients with recurrent FL were treated with ibrutinib 560 mg/d until progression or intolerance. The primary end point was overall response rate (ORR). Exploratory analyses included correlations of outcome with recurrent mutations identified in a cancer gene panel that used next-generation sequencing in pretreatment biopsies from 31 patients and results of early interim positron emission tomography/computed tomography scans in 20 patients. ORR was 37.5% with a complete response rate of 12.5%, median progression-free survival (PFS) of 14 months, and 2-year PFS of 20.4%. Response rates were significantly higher among patients whose disease was sensitive to rituximab (52.6%) compared with those who were rituximab refractory (16.7%) (P = .04). CARD11 mutations were present in 16% of patients (5 of 31) and predicted resistance to ibrutinib with only wild-type patients responding (P = .002). Maximum standardized uptake value at cycle 1 day 8 correlated with response and PFS. Ibrutinib was well-tolerated with a toxicity profile similar to labeled indications. Ibrutinib is a well-tolerated treatment with modest activity in relapsed FL. Evaluation of BTK inhibitors in earlier lines of therapy may be warranted on the basis of improved response rates in rituximab-sensitive disease. Somatic mutations such as CARD11 may have an impact on response to ibrutinib, may inform clinical decisions, and should be evaluated in larger data sets. This trial was registered at www.clinicaltrials.gov as #NCT01849263.
Assuntos
Tirosina Quinase da Agamaglobulinemia/antagonistas & inibidores , Linfoma Folicular/tratamento farmacológico , Recidiva Local de Neoplasia/tratamento farmacológico , Inibidores de Proteínas Quinases/uso terapêutico , Pirazóis/uso terapêutico , Pirimidinas/uso terapêutico , Adenina/análogos & derivados , Idoso , Idoso de 80 Anos ou mais , Proteínas Adaptadoras de Sinalização CARD/genética , Progressão da Doença , Feminino , Guanilato Ciclase/genética , Humanos , Linfoma Folicular/genética , Linfoma Folicular/patologia , Masculino , Pessoa de Meia-Idade , Mutação , Recidiva Local de Neoplasia/genética , Recidiva Local de Neoplasia/patologia , Piperidinas , Inibidores de Proteínas Quinases/administração & dosagem , Inibidores de Proteínas Quinases/efeitos adversos , Pirazóis/administração & dosagem , Pirazóis/efeitos adversos , Pirimidinas/administração & dosagem , Pirimidinas/efeitos adversos , Resultado do TratamentoRESUMO
PURPOSE: Following automated variant calling, manual review of aligned read sequences is required to identify a high-quality list of somatic variants. Despite widespread use in analyzing sequence data, methods to standardize manual review have not been described, resulting in high inter- and intralab variability. METHODS: This manual review standard operating procedure (SOP) consists of methods to annotate variants with four different calls and 19 tags. The calls indicate a reviewer's confidence in each variant and the tags indicate commonly observed sequencing patterns and artifacts that inform the manual review call. Four individuals were asked to classify variants prior to, and after, reading the SOP and accuracy was assessed by comparing reviewer calls with orthogonal validation sequencing. RESULTS: After reading the SOP, average accuracy in somatic variant identification increased by 16.7% (p value = 0.0298) and average interreviewer agreement increased by 12.7% (p value < 0.001). Manual review conducted after reading the SOP did not significantly increase reviewer time. CONCLUSION: This SOP supports and enhances manual somatic variant detection by improving reviewer accuracy while reducing the interreviewer variability for variant calling and annotation.
Assuntos
Sequenciamento de Nucleotídeos em Larga Escala/normas , Mutação/genética , Neoplasias/genética , Software , Algoritmos , Humanos , Neoplasias/patologia , Polimorfismo de Nucleotídeo Único/genética , Alinhamento de SequênciaRESUMO
Synaptic plasticities, such as long-term potentiation (LTP) and depression (LTD), tune synaptic efficacy and are essential for learning and memory. Current studies of synaptic plasticity in humans are limited by a lack of adequate human models. Here, we modeled the thalamocortical system by fusing human induced pluripotent stem cell-derived thalamic and cortical organoids. Single-nucleus RNA sequencing revealed that >80% of cells in thalamic organoids were glutamatergic neurons. When fused to form thalamocortical assembloids, thalamic and cortical organoids formed reciprocal long-range axonal projections and reciprocal synapses detectable by light and electron microscopy, respectively. Using whole-cell patch-clamp electrophysiology and two-photon imaging, we characterized glutamatergic synaptic transmission. Thalamocortical and corticothalamic synapses displayed short-term plasticity analogous to that in animal models. LTP and LTD were reliably induced at both synapses; however, their mechanisms differed from those previously described in rodents. Thus, thalamocortical assembloids provide a model system for exploring synaptic plasticity in human circuits.
RESUMO
Synaptic plasticities, such as long-term potentiation (LTP) and depression (LTD), tune synaptic efficacy and are essential for learning and memory. Current studies of synaptic plasticity in humans are limited by a lack of adequate human models. Here, we modeled the thalamocortical system by fusing human induced pluripotent stem cell-derived thalamic and cortical organoids. Single-nucleus RNA-sequencing revealed that most cells in mature thalamic organoids were glutamatergic neurons. When fused to form thalamocortical assembloids, thalamic and cortical organoids formed reciprocal long-range axonal projections and reciprocal synapses detectable by light and electron microscopy, respectively. Using whole-cell patch-clamp electrophysiology and two-photon imaging, we characterized glutamatergic synaptic transmission. Thalamocortical and corticothalamic synapses displayed short-term plasticity analogous to that in animal models. LTP and LTD were reliably induced at both synapses; however, their mechanisms differed from those previously described in rodents. Thus, thalamocortical assembloids provide a model system for exploring synaptic plasticity in human circuits.
RESUMO
ABSTRACT: Personalized cancer vaccines designed to target neoantigens represent a promising new treatment paradigm in oncology. In contrast to classical idiotype vaccines, we hypothesized that "polyvalent" vaccines could be engineered for the personalized treatment of follicular lymphoma (FL) using neoantigen discovery by combined whole-exome sequencing (WES) and RNA sequencing (RNA-seq). Fifty-eight tumor samples from 57 patients with FL underwent WES and RNA-seq. Somatic and B-cell clonotype neoantigens were predicted and filtered to identify high-quality neoantigens. B-cell clonality was determined by the alignment of B-cell receptor (BCR) CDR3 regions from RNA-seq data, grouping at the protein level, and comparison with the BCR repertoire from healthy individuals using RNA-seq data. An average of 52 somatic mutations per patient (range, 2-172) were identified, and ≥2 (median, 15) high-quality neoantigens were predicted for 56 of 58 FL samples. The predicted neoantigen peptides were composed of missense mutations (77%), indels (9%), gene fusions (3%), and BCR sequences (11%). Building off of these preclinical analyses, we initiated a pilot clinical trial using personalized neoantigen vaccination combined with PD-1 blockade in patients with relapsed or refractory FL (#NCT03121677). Synthetic long peptide vaccines targeting predicted high-quality neoantigens were successfully synthesized for and administered to all 4 patients enrolled. Initial results demonstrate feasibility, safety, and potential immunologic and clinical responses. Our study suggests that a genomics-driven personalized cancer vaccine strategy is feasible for patients with FL, and this may overcome prior challenges in the field. This trial was registered at www.ClinicalTrials.gov as #NCT03121677.
Assuntos
Antígenos de Neoplasias , Vacinas Anticâncer , Linfoma Folicular , Medicina de Precisão , Humanos , Linfoma Folicular/terapia , Linfoma Folicular/imunologia , Linfoma Folicular/genética , Vacinas Anticâncer/imunologia , Vacinas Anticâncer/uso terapêutico , Antígenos de Neoplasias/imunologia , Medicina de Precisão/métodos , Pessoa de Meia-Idade , Feminino , Masculino , Idoso , Adulto , Sequenciamento do Exoma , MutaçãoRESUMO
Previous studies have demonstrated the dynamic changes in chromatin structure during retinal development that correlate with changes in gene expression. However, a major limitation of those prior studies was the lack of cellular resolution. Here, we integrate single-cell (sc) RNA-seq and scATAC-seq with bulk retinal data sets to identify cell type-specific changes in the chromatin structure during development. Although most genes' promoter activity is strongly correlated with chromatin accessibility, we discovered several hundred genes that were transcriptionally silent but had accessible chromatin at their promoters. Most of those silent/accessible gene promoters were in the Müller glial cells. The Müller cells are radial glia of the retina and perform a variety of essential functions to maintain retinal homeostasis and respond to stress, injury, or disease. The silent/accessible genes in Müller glia are enriched in pathways related to inflammation, angiogenesis, and other types of cell-cell signaling and were rapidly activated when we tested 15 different physiologically relevant conditions to mimic retinal stress, injury, or disease in human and murine retinae. We refer to these as "pliancy genes" because they allow the Müller glia to rapidly change their gene expression and cellular state in response to different types of retinal insults. The Müller glial cell pliancy program is established during development, and we demonstrate that pliancy genes are necessary and sufficient for regulating inflammation in the murine retina in vivo. In zebrafish, Müller glia can de-differentiate and form retinal progenitor cells that replace lost neurons. The pro-inflammatory pliancy gene cascade is not activated in zebrafish Müller glia following injury, and we propose a model in which species-specific pliancy programs underly the differential response to retinal damage in species that can regenerate retinal neurons (zebrafish) versus those that cannot (humans and mice).
RESUMO
Super-enhancers (SEs) are expansive regions of genomic DNA that regulate the expression of genes involved in cell identity and cell fate. Recently, we found that distinct modules within a murine SE regulate gene expression of master regulatory transcription factor Vsx2 in a developmental stage- and cell-type specific manner. Vsx2 is expressed in retinal progenitor cells as well as differentiated bipolar neurons and Müller glia. Mutations in VSX2 in humans and mice lead to microphthalmia due to a defect in retinal progenitor cell proliferation. Deletion of a single module within the Vsx2 SE leads to microphthalmia. Deletion of a separate module within the SE leads to a complete loss of bipolar neurons, yet the remainder of the retina develops normally. Furthermore, the Vsx2 SE is evolutionarily conserved in vertebrates, suggesting that these modules are important for retinal development across species. In the present study, we examine the ability of these modules to drive retinal development between species. By inserting the human build of one Vsx2 SE module into a mouse with microphthalmia, eye size was rescued. To understand the implications of these SE modules in a model of human development, we generated human retinal organoids. Deleting one module results in small organoids, recapitulating the small-eyed phenotype of mice with microphthalmia, while deletion of the other module leads to a complete loss of ON cone bipolar neurons. This prototypical SE serves as a model for uncoupling developmental stage- and cell-type specific effects of neurogenic transcription factors with complex expression patterns. Moreover, by elucidating the gene regulatory mechanisms, we can begin to examine how dysregulation of these mechanisms contributes to phenotypic diversity and disease.
RESUMO
Neoantigens are tumor-specific peptide sequences resulting from sources such as somatic DNA mutations. Upon loading onto major histocompatibility complex (MHC) molecules, they can trigger recognition by T cells. Accurate neoantigen identification is thus critical for both designing cancer vaccines and predicting response to immunotherapies. Neoantigen identification and prioritization relies on correctly predicting whether the presenting peptide sequence can successfully induce an immune response. Because most somatic mutations are single-nucleotide variants, changes between wild-type and mutated peptides are typically subtle and require cautious interpretation. A potentially underappreciated variable in neoantigen prediction pipelines is the mutation position within the peptide relative to its anchor positions for the patient's specific MHC molecules. Whereas a subset of peptide positions are presented to the T cell receptor for recognition, others are responsible for anchoring to the MHC, making these positional considerations critical for predicting T cell responses. We computationally predicted anchor positions for different peptide lengths for 328 common HLA alleles and identified unique anchoring patterns among them. Analysis of 923 tumor samples shows that 6 to 38% of neoantigen candidates are potentially misclassified and can be rescued using allele-specific knowledge of anchor positions. A subset of anchor results were orthogonally validated using protein crystallography structures. Representative anchor trends were experimentally validated using peptide-MHC stability assays and competition binding assays. By incorporating our anchor prediction results into neoantigen prediction pipelines, we hope to formalize, streamline, and improve the identification process for relevant clinical studies.
Assuntos
Antígenos de Neoplasias , Neoplasias , Humanos , Antígenos de Neoplasias/genética , Linfócitos T , Mutação , Peptídeos/genéticaRESUMO
Follicular lymphoma (FL) is clinically heterogeneous, with select patients tolerating extended watch-and-wait, whereas others require prompt treatment, suffer progression of disease within 24 months of treatment (POD24), and/or experience aggressive histologic transformation (t-FL). Because our understanding of the relationship between genetic alterations in FL and patient outcomes remains limited, we conducted a clinicogenomic analysis of 370 patients with FL or t-FL (from Cancer and Leukemia Group B/Alliance trials 50402/50701/50803, or real-world cohorts from Washington University School of Medicine, Cleveland Clinic, or University of Miami). FL subsets by grade, stage, watch-and-wait, or POD24 status did not differ by mutation burden, whereas mutation burden was significantly higher in relapsed/refractory (rel/ref) FL and t-FL than in newly diagnosed (dx) FL. Nonetheless, mutation burden in dx FL was not associated with frontline progression-free survival (PFS). CREBBP was the only gene more commonly mutated in FL than in t-FL yet mutated CREBBP was associated with shorter frontline PFS in FL. Mutations in 20 genes were more common in rel/ref FL or t-FL than in dx FL, including 6 significantly mutated genes (SMGs): STAT6, TP53, IGLL5, B2M, SOCS1, and MYD88. We defined a mutations associated with progression (MAP) signature as ≥2 mutations in these 7 genes (6 rel/ref FL or t-FL SMGs plus CREBBP). Patients with dx FL possessing a MAP signature had shorter frontline PFS, revealing a 7-gene set offering insight into FL progression risk potentially more generalizable than the m7-Follicular Lymphoma International Prognostic Index (m7-FLIPI), which had modest prognostic value in our cohort. Future studies are warranted to validate the poor prognosis associated with a MAP signature in dx FL, potentially facilitating novel trials specifically in this high-risk subset of patients.
Assuntos
Linfoma Folicular , Humanos , Linfoma Folicular/diagnóstico , Linfoma Folicular/genética , Fatores de Risco , Prognóstico , Intervalo Livre de Progressão , MutaçãoRESUMO
The malignant Hodgkin and Reed Sternberg (HRS) cells of classical Hodgkin lymphoma (cHL) are scarce in affected lymph nodes, creating a challenge to detect driver somatic mutations. As an alternative to cell purification techniques, we hypothesized that ultra-deep exome sequencing would allow genomic study of HRS cells, thereby streamlining analysis and avoiding technical pitfalls. To test this, 31 cHL tumor/normal pairs were exome sequenced to approximately 1,000× median depth of coverage. An orthogonal error-corrected sequencing approach verified >95% of the discovered mutations. We identified mutations in genes novel to cHL including: CDH5 and PCDH7, novel stop gain mutations in IL4R, and a novel pattern of recurrent mutations in pathways regulating Hippo signaling. As a further application of our exome sequencing, we attempted to identify expressed somatic single-nucleotide variants (SNV) in single-nuclei RNA sequencing (snRNA-seq) data generated from a patient in our cohort. Our snRNA analysis identified a clear cluster of cells containing a somatic SNV identified in our deep exome data. This cluster has differentially expressed genes that are consistent with genes known to be dysregulated in HRS cells (e.g., PIM1 and PIM3). The cluster also contains cells with an expanded B-cell clonotype further supporting a malignant phenotype. This study provides proof-of-principle that ultra-deep exome sequencing can be utilized to identify recurrent mutations in HRS cells and demonstrates the feasibility of snRNA-seq in the context of cHL. These studies provide the foundation for the further analysis of genomic variants in large cohorts of patients with cHL. SIGNIFICANCE: Our data demonstrate the utility of ultra-deep exome sequencing in uncovering somatic variants in Hodgkin lymphoma, creating new opportunities to define the genes that are recurrently mutated in this disease. We also show for the first time the successful application of snRNA-seq in Hodgkin lymphoma and describe the expression profile of a putative cluster of HRS cells in a single patient.
Assuntos
Doença de Hodgkin , Humanos , Doença de Hodgkin/genética , Células de Reed-Sternberg/metabolismo , Mutação/genética , Sequenciamento de Nucleotídeos em Larga Escala , RNA Nuclear Pequeno/metabolismoRESUMO
Super-enhancers are expansive regions of genomic DNA comprised of multiple putative enhancers that contribute to the dynamic gene expression patterns during development. This is particularly important in neurogenesis because many essential transcription factors have complex developmental stage- and cell-type specific expression patterns across the central nervous system. In the developing retina, Vsx2 is expressed in retinal progenitor cells and is maintained in differentiated bipolar neurons and Müller glia. A single super-enhancer controls this complex and dynamic pattern of expression. Here we show that deletion of one region disrupts retinal progenitor cell proliferation but does not affect cell fate specification. The deletion of another region has no effect on retinal progenitor cell proliferation but instead leads to a complete loss of bipolar neurons. This prototypical super-enhancer may serve as a model for dissecting the complex gene expression patterns for neurogenic transcription factors during development. Moreover, it provides a unique opportunity to alter expression of individual transcription factors in particular cell types at specific stages of development. This provides a deeper understanding of function that cannot be achieved with traditional knockout mouse approaches.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Neurogênese/fisiologia , Sequências Reguladoras de Ácido Nucleico , Retina/metabolismo , Animais , Sistemas CRISPR-Cas , Diferenciação Celular/genética , Proliferação de Células , Epigenômica , Feminino , Proteínas de Homeodomínio/química , Proteínas de Homeodomínio/genética , Masculino , Camundongos , Neurogênese/genética , Neuroglia/fisiologia , Neurônios/metabolismo , Sequências Reguladoras de Ácido Nucleico/genética , Células-Tronco/fisiologia , Fatores de Transcrição/química , Fatores de Transcrição/fisiologiaRESUMO
INTRODUCTION: Recent advances have led to the design of a new cephalomedullary nail, which aims to decrease the risk of failures in patients with intertrochanteric hip fractures by allowing for insertion of two interdigitating screws into the head segment. The goal of this study is to evaluate the safety and efficacy of this two-screw cephalomedullary nailing system. PATIENTS/PARTICIPANTS: Patients 18 years of age and older who underwent intramedullary nailing of their intertrochanteric femoral fracture using the InterTAN nailing system (Smith and Nephew, Memphis, TN) from 2012 to 2016 were included in this retrospective study which was performed at two urban certified level-1 trauma centers and one urban certified level-3 trauma center. The study data was collected through a retrospective chart review and review of the existing radiographic studies. Primary outcome measure was mechanical hardware failure and screw cutout. Secondary outcome measures included nonunion, malunion, medical and surgical complications. RESULTS: A total of 264 patients were included in this analysis. Two patients (0.75%) were found to have a screw cut out requiring revision surgery. Two other revision surgeries were performed for malrotation (n = 1) and malunion (n = 1). Other implant-related complications occurred in 19 cases (7.9%), which included broken distal screws (n = 9), distal screw loosening (n = 8), and loose lag screws (n = 2). There was a total of 10 (3.8%) surgical wound complications, including four deep and six superficial infections. DISCUSSION: This modified cephalomedullary nail is a reliable, safe, and effective implant for management of intertrochanteric hip fractures. Surgical treatment of patients with intertrochanteric hip fractures can be performed in a safe fashion using this implant.