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1.
Radiology ; 294(2): 421-431, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31793848

RESUMO

BackgroundDeep learning has the potential to augment the use of chest radiography in clinical radiology, but challenges include poor generalizability, spectrum bias, and difficulty comparing across studies.PurposeTo develop and evaluate deep learning models for chest radiograph interpretation by using radiologist-adjudicated reference standards.Materials and MethodsDeep learning models were developed to detect four findings (pneumothorax, opacity, nodule or mass, and fracture) on frontal chest radiographs. This retrospective study used two data sets. Data set 1 (DS1) consisted of 759 611 images from a multicity hospital network and ChestX-ray14 is a publicly available data set with 112 120 images. Natural language processing and expert review of a subset of images provided labels for 657 954 training images. Test sets consisted of 1818 and 1962 images from DS1 and ChestX-ray14, respectively. Reference standards were defined by radiologist-adjudicated image review. Performance was evaluated by area under the receiver operating characteristic curve analysis, sensitivity, specificity, and positive predictive value. Four radiologists reviewed test set images for performance comparison. Inverse probability weighting was applied to DS1 to account for positive radiograph enrichment and estimate population-level performance.ResultsIn DS1, population-adjusted areas under the receiver operating characteristic curve for pneumothorax, nodule or mass, airspace opacity, and fracture were, respectively, 0.95 (95% confidence interval [CI]: 0.91, 0.99), 0.72 (95% CI: 0.66, 0.77), 0.91 (95% CI: 0.88, 0.93), and 0.86 (95% CI: 0.79, 0.92). With ChestX-ray14, areas under the receiver operating characteristic curve were 0.94 (95% CI: 0.93, 0.96), 0.91 (95% CI: 0.89, 0.93), 0.94 (95% CI: 0.93, 0.95), and 0.81 (95% CI: 0.75, 0.86), respectively.ConclusionExpert-level models for detecting clinically relevant chest radiograph findings were developed for this study by using adjudicated reference standards and with population-level performance estimation. Radiologist-adjudicated labels for 2412 ChestX-ray14 validation set images and 1962 test set images are provided.© RSNA, 2019Online supplemental material is available for this article.See also the editorial by Chang in this issue.

2.
Adv Exp Med Biol ; 1168: 103-115, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31713167

RESUMO

The past two decades have seen unprecedented advances in the field of oncogenomics. The ongoing characterization of neoplastic tissues through genomic techniques has transformed many aspects of cancer research, diagnosis, and treatment. However, identifying sequence variants with biological and clinical significance is a challenging endeavor. In order to accomplish this task, variants must be annotated and interpreted using various online resources. Data on protein structure, functional prediction, variant frequency in relevant populations, and multiple other factors have been compiled in useful databases for this purpose. Thus, understanding the available online resources for the annotation and interpretation of sequence variants is critical to aid molecular pathologists and researchers working in this space.


Assuntos
Bases de Dados Genéticas , Privacidade Genética , Neoplasias , Farmacogenética , Privacidade Genética/tendências , Variação Genética , Recursos em Saúde , Humanos , Internet , Neoplasias/fisiopatologia , Neoplasias/terapia , Análise de Sequência de DNA/normas , Análise de Sequência de DNA/tendências
3.
Appl Immunohistochem Mol Morphol ; 27(10): 740-748, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31702703

RESUMO

Subcutaneous panniculitis-like T-cell lymphoma (SPTCL) is a malignant primary cutaneous T-cell lymphoma that is challenging to distinguish from other neoplastic and reactive panniculitides. In an attempt to identify somatic variants in SPTCL that may be diagnostically or therapeutically relevant, we performed both exome sequencing on paired tumor-normal samples and targeted sequencing of hematolymphoid-malignancy-associated genes on tumor biopsies. Exome sequencing was performed on skin biopsies from 4 cases of skin-limited SPTCL, 1 case of peripheral T-cell lymphoma, not otherwise specified with secondary involvement of the panniculus, and 2 cases of lupus panniculitis. This approach detected between 1 and 13 high-confidence somatic variants that were predicted to result in a protein alteration per case. Variants of interest identified include 1 missense mutation in ARID1B in 1 case of SPTCL. To detect variants that were present at a lower level, we used a more sensitive targeted panel to sequence 41 hematolymphoid-malignancy-associated genes. The targeted panel was applied to 2 of the biopsies that were evaluated by whole exome sequencing as well as 5 additional biopsies. Potentially pathogenic variants were identified in KMT2D and PLCG1 among others, but no gene was altered in >2 of the 7 cases sequenced. One variant that was notably absent from the cases sequences is RHOA G17V. Further work will be required to further elucidate the genetic abnormalities that lead to this rare lymphoma.

4.
Int J Gynecol Pathol ; 38(4): 386-392, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-29620581

RESUMO

Low-grade serous carcinomas only rarely coexist with or progress to high-grade tumors. We present a case of low-grade serous carcinoma with transformation to carcinosarcoma on recurrence in the lymph node. Identical BRAF V600E and telomerase reverse transcriptase promoter mutations were identified in both the original and recurrent tumor. Given that telomerase reverse transcriptase promotor mutations are thought to play a role in progression of other tumor types, the function of telomerase reverse transcriptase mutations in BRAF mutated low-grade serous carcinoma deserves investigation.


Assuntos
Carcinossarcoma/diagnóstico , Neoplasias Ovarianas/diagnóstico , Regiões Promotoras Genéticas/genética , Proteínas Proto-Oncogênicas B-raf/genética , Telomerase/genética , Idoso , Carcinossarcoma/genética , Carcinossarcoma/patologia , Progressão da Doença , Feminino , Humanos , Linfonodos/patologia , Mutação , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/patologia , Ovário/patologia
5.
Am J Surg Pathol ; 42(12): 1636-1646, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30312179

RESUMO

Advances in the quality of whole-slide images have set the stage for the clinical use of digital images in anatomic pathology. Along with advances in computer image analysis, this raises the possibility for computer-assisted diagnostics in pathology to improve histopathologic interpretation and clinical care. To evaluate the potential impact of digital assistance on interpretation of digitized slides, we conducted a multireader multicase study utilizing our deep learning algorithm for the detection of breast cancer metastasis in lymph nodes. Six pathologists reviewed 70 digitized slides from lymph node sections in 2 reader modes, unassisted and assisted, with a wash-out period between sessions. In the assisted mode, the deep learning algorithm was used to identify and outline regions with high likelihood of containing tumor. Algorithm-assisted pathologists demonstrated higher accuracy than either the algorithm or the pathologist alone. In particular, algorithm assistance significantly increased the sensitivity of detection for micrometastases (91% vs. 83%, P=0.02). In addition, average review time per image was significantly shorter with assistance than without assistance for both micrometastases (61 vs. 116 s, P=0.002) and negative images (111 vs. 137 s, P=0.018). Lastly, pathologists were asked to provide a numeric score regarding the difficulty of each image classification. On the basis of this score, pathologists considered the image review of micrometastases to be significantly easier when interpreted with assistance (P=0.0005). Utilizing a proof of concept assistant tool, this study demonstrates the potential of a deep learning algorithm to improve pathologist accuracy and efficiency in a digital pathology workflow.


Assuntos
Neoplasias da Mama/patologia , Aprendizado Profundo , Diagnóstico por Computador/métodos , Interpretação de Imagem Assistida por Computador/métodos , Linfonodos/patologia , Patologia Clínica/métodos , Biópsia , Feminino , Humanos , Metástase Linfática , Micrometástase de Neoplasia , Variações Dependentes do Observador , Reconhecimento Automatizado de Padrão , Valor Preditivo dos Testes , Estudo de Prova de Conceito , Reprodutibilidade dos Testes , Fatores de Tempo , Fluxo de Trabalho
6.
Methods Mol Biol ; 1799: 341-351, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29956162

RESUMO

Transgenic methods to manipulate CD4 T lymphocytes in vivo via forced expression of TCR transgenes and targeted "knockout" of individual genes by Cre-lox technology are fundamental to modern immunology. However, efforts to scale up functional analysis by modifying expression of larger numbers of genes in T cells ex vivo have proven surprisingly difficult. Early RNA interference experiments achieved successful small RNA transfection by using very high concentrations of short-interfering RNA (siRNA) [1], but primary T cells are generally resistant to standard electroporation, cationic liposome-, and calcium phosphate-mediated transfection methods. Moreover, although viral vectors can successfully introduce DNA fragments of varying length, expression of these constructs in primary T cells is low efficiency and the subcloning process laborious. In this context, the relatively recent discovery of dozens of highly expressed microRNAs (miRNAs) in the immune system provides both an opportunity and a new challenge [2, 3]. How can we query the miRNAome of a cell to assign particular roles to individual miRNAs? Here, we describe an optimized technique for efficient and reproducible transfection of primary mouse CD4 T cells in vitro with synthetic miRNA mimics.


Assuntos
Hipersensibilidade/genética , MicroRNAs/genética , Animais , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Eletroporação , Expressão Gênica , Hipersensibilidade/imunologia , Ativação Linfocitária , Camundongos , Linfócitos T/imunologia , Linfócitos T/metabolismo
7.
Immunity ; 44(4): 821-32, 2016 Apr 19.
Artigo em Inglês | MEDLINE | ID: mdl-26850657

RESUMO

MicroRNAs (miRNAs) are important regulators of cell fate decisions in immune responses. They act by coordinate repression of multiple target genes, a property that we exploited to uncover regulatory networks that govern T helper-2 (Th2) cells. A functional screen of individual miRNAs in primary T cells uncovered multiple miRNAs that inhibited Th2 cell differentiation. Among these were miR-24 and miR-27, miRNAs coexpressed from two genomic clusters, which each functioned independently to limit interleukin-4 (IL-4) production. Mice lacking both clusters in T cells displayed increased Th2 cell responses and tissue pathology in a mouse model of asthma. Gene expression and pathway analyses placed miR-27 upstream of genes known to regulate Th2 cells. They also identified targets not previously associated with Th2 cell biology which regulated IL-4 production in unbiased functional testing. Thus, elucidating the biological function and target repertoire of miR-24 and miR-27 reveals regulators of Th2 cell biology.


Assuntos
Asma/imunologia , Interleucina-4/biossíntese , MicroRNAs/genética , Células Th2/imunologia , Animais , Sequência de Bases , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Células Cultivadas , Modelos Animais de Doenças , Feminino , Inflamação/imunologia , Interleucina-4/imunologia , Ativação Linfocitária/imunologia , Masculino , Camundongos , Camundongos Knockout , Família Multigênica/genética , Análise de Sequência de RNA , Células Th2/citologia
8.
Immunity ; 35(2): 169-81, 2011 Aug 26.
Artigo em Inglês | MEDLINE | ID: mdl-21820330

RESUMO

MicroRNA (miRNA)-deficient helper T cells exhibit abnormal IFN-γ production and decreased proliferation. However, the contributions of individual miRNAs to this phenotype remain poorly understood. We conducted a screen for miRNA function in primary T cells and identified individual miRNAs that rescue the defects associated with miRNA deficiency. Multiple members of the miR-17 and miR-92 families enhanced miRNA-deficient T cell proliferation whereas miR-29 largely corrected their aberrant interferon-γ (IFN-γ) expression. Repression of IFN-γ production by miR-29 involved direct targeting of both T-bet and Eomes, two transcription factors known to induce IFN-γ production. Although not usually expressed at functionally relevant amounts in helper T cells, Eomes was abundant in miRNA-deficient cells and was upregulated after miR-29 inhibition in wild-type cells. These results demonstrate that miR-29 regulates helper T cell differentiation by repressing multiple target genes, including at least two that are independently capable of inducing the T helper 1 (Th1) cell gene expression program.


Assuntos
Interferon gama/metabolismo , MicroRNAs/metabolismo , Proteínas com Domínio T/metabolismo , Linfócitos T Auxiliares-Indutores/metabolismo , Animais , Diferenciação Celular , Proliferação de Células , Células Cultivadas , Citocinas/genética , Citocinas/imunologia , Citocinas/metabolismo , Regulação da Expressão Gênica/imunologia , Interferon gama/genética , Interferon gama/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , MicroRNAs/genética , MicroRNAs/imunologia , Proteínas/genética , Proteínas de Ligação a RNA , Proteínas com Domínio T/imunologia , Linfócitos T Auxiliares-Indutores/citologia , Linfócitos T Auxiliares-Indutores/imunologia
9.
J Immunol ; 185(7): 3835-46, 2010 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-20805417

RESUMO

MicroRNAs (miRNAs) are small noncoding RNAs that have recently emerged as critical regulators of gene expression within the immune system. In this study, we used mice with conditional deletion of Dicer and DiGeorge syndrome critical region 8 (Dgcr8) to dissect the roles of miRNAs in NK cell activation, survival, and function during viral infection. We developed a system for deletion of either Dicer or Dgcr8 in peripheral NK cells via drug-induced Cre activity. We found that Dicer- and Dgcr8-deficient NK cells were significantly impaired in survival and turnover, and had impaired function of the ITAM-containing activating NK cell receptors. We further demonstrated that both Dicer- and Dgcr8-dependent pathways were indispensable for the expansion of Ly49H(+) NK cells during mouse cytomegalovirus infection. Our data indicate similar phenotypes for Dicer- and Dgcr8-deficient NK cells, which strongly suggest that these processes are regulated by miRNAs. Thus, our findings indicate a critical role for miRNAs in controlling NK cell homeostasis and effector function, with implications for miRNAs regulating diverse aspects of NK cell biology.


Assuntos
Perfilação da Expressão Gênica , Células Matadoras Naturais/metabolismo , Ativação Linfocitária/genética , MicroRNAs/metabolismo , Transferência Adotiva , Animais , Separação Celular , Sobrevivência Celular , RNA Helicases DEAD-box/genética , Endorribonucleases/genética , Citometria de Fluxo , Expressão Gênica , Técnicas de Inativação de Genes , Humanos , Células Matadoras Naturais/imunologia , Ativação Linfocitária/imunologia , Camundongos , Camundongos Endogâmicos C57BL , MicroRNAs/genética , Análise de Sequência com Séries de Oligonucleotídeos , Proteínas/genética , Proteínas de Ligação a RNA , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Ribonuclease III
10.
J Biol Chem ; 281(11): 7445-51, 2006 Mar 17.
Artigo em Inglês | MEDLINE | ID: mdl-16407207

RESUMO

The binding of the RNA polymerase III (pol III) transcription factor TFIIIC to the box A intragenic promoter element of tRNA genes specifies the placement of TFIIIB on upstream-lying DNA. In turn, TFIIIB recruits pol III to the promoter and specifies transcription initiating 17-19 base pairs upstream of box A. The resolution of the pol III transcription apparatus into recombinant TFIIIB, highly purified TFIIIC, and pol III is accompanied by a loss of precision in specifying where transcription initiation occurs due to heterogeneous placement of TFIIIB. In this paper we show that Nhp6a, an abundant high mobility group B (HMGB) family, non-sequence-specific DNA-binding protein in Saccharomyces cerevisiae restores transcriptional initiation fidelity to this highly purified in vitro system. Restoration of initiation fidelity requires the presence of Nhp6a prior to TFIIIB-DNA complex formation. Chemical nuclease footprinting of TFIIIC- and TFIIIB-TFIIIC-DNA complexes reveals that Nhp6a markedly alters the TFIIIC footprint over box A and reduces the size of the TFIIIB footprint on upstream DNA sequence. Analyses of unprocessed tRNAs from yeast lacking Nhp6a and its closely related paralogue Nhp6b demonstrate that Nhp6 is required for transcriptional initiation fidelity of some but not all tRNA genes, in vivo.


Assuntos
Proteínas de Ligação a DNA/fisiologia , Regulação Fúngica da Expressão Gênica , Proteínas Nucleares/fisiologia , RNA Polimerase III/química , Proteínas de Saccharomyces cerevisiae/fisiologia , Fator de Transcrição TFIIIB/química , Fatores de Transcrição TFIII/química , Sequência de Bases , Sítios de Ligação , DNA/química , Proteínas de Ligação a DNA/metabolismo , Proteínas HMGN , Técnicas In Vitro , Íntrons , Dados de Sequência Molecular , Proteínas Nucleares/metabolismo , Plasmídeos/metabolismo , Regiões Promotoras Genéticas , Ligação Proteica , RNA de Transferência/química , RNA de Transferência/metabolismo , Proteínas Recombinantes/química , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Temperatura Ambiente , Transcrição Genética
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