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1.
J Pathol ; 248(2): 164-178, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30690729

RESUMO

Combined hepatocellular-cholangiocarcinomas (CHC) are mixed tumours with both hepatocellular carcinoma (HCC) and cholangiocarcinoma (CC) components. CHC prognosis is similar to intrahepatic CC (ICC) and worse than HCC; staging and treatment generally follow ICC algorithms. However, the molecular biology of CHC remains poorly characterised. We performed capture-based next-generation sequencing of 20 CHC and, for comparison, 10 ICC arising in cirrhosis. Intratumour heterogeneity was assessed by separately sequencing the HCC and CC components of nine CHC. CHC demonstrated molecular profiles similar to HCC, even in the CC component. CHC harboured recurrent alterations in TERT (80%), TP53 (80%), cell cycle genes (40%; CCND1, CCNE1, CDKN2A), receptor tyrosine kinase/Ras/PI3-kinase pathway genes (55%; MET, ERBB2, KRAS, PTEN), chromatin regulators (20%; ARID1A, ARID2) and Wnt pathway genes (20%; CTNNB1, AXIN, APC). No CHC had alterations in IDH1, IDH2, FGFR2 or BAP1, genes typically mutated in ICC. TERT promoter mutations were consistently identified in both HCC and CC components, supporting TERT alteration as an early event in CHC evolution. TP53 mutations were present in both components in slightly over half the TP53-altered cases. By contrast, focal amplifications of CCND1, MET and ERRB2, as well as Wnt pathway alterations, were most often exclusive to one component, suggesting that these are late events in CHC evolution. ICC in cirrhosis demonstrated alterations similar to ICC in non-cirrhotic liver, including in IDH1 or IDH2 (30%), CDKN2A (40%), FGFR2 (20%), PBRM1 (20%), ARID1A (10%) and BAP1 (10%). TERT promoter and TP53 mutation were present in only one ICC each. Our data demonstrate that CHC genetics are distinct from ICC (even in cirrhosis) and similar to HCC, which has diagnostic utility and implications for treatment. Copyright © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.


Assuntos
Biomarcadores Tumorais/genética , Carcinoma Hepatocelular/genética , Colangiocarcinoma/genética , Perfilação da Expressão Gênica , Neoplasias Hepáticas/genética , Neoplasias Complexas Mistas/genética , Transcriptoma , Adulto , Idoso , Carcinoma Hepatocelular/patologia , Colangiocarcinoma/patologia , Feminino , Dosagem de Genes , Rearranjo Gênico , Predisposição Genética para Doença , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Neoplasias Hepáticas/patologia , Masculino , Pessoa de Meia-Idade , Mutação , Neoplasias Complexas Mistas/patologia
2.
N Engl J Med ; 373(20): 1926-36, 2015 Nov 12.
Artigo em Inglês | MEDLINE | ID: mdl-26559571

RESUMO

BACKGROUND: The pathogenic mutations in melanoma have been largely catalogued; however, the order of their occurrence is not known. METHODS: We sequenced 293 cancer-relevant genes in 150 areas of 37 primary melanomas and their adjacent precursor lesions. The histopathological spectrum of these areas included unequivocally benign lesions, intermediate lesions, and intraepidermal or invasive melanomas. RESULTS: Precursor lesions were initiated by mutations of genes that are known to activate the mitogen-activated protein kinase pathway. Unequivocally benign lesions harbored BRAF V600E mutations exclusively, whereas those categorized as intermediate were enriched for NRAS mutations and additional driver mutations. A total of 77% of areas of intermediate lesions and melanomas in situ harbored TERT promoter mutations, a finding that indicates that these mutations are selected at an unexpectedly early stage of the neoplastic progression. Biallelic inactivation of CDKN2A emerged exclusively in invasive melanomas. PTEN and TP53 mutations were found only in advanced primary melanomas. The point-mutation burden increased from benign through intermediate lesions to melanoma, with a strong signature of the effects of ultraviolet radiation detectable at all evolutionary stages. Copy-number alterations became prevalent only in invasive melanomas. Tumor heterogeneity became apparent in the form of genetically distinct subpopulations as melanomas progressed. CONCLUSIONS: Our study defined the succession of genetic alterations during melanoma progression, showing distinct evolutionary trajectories for different melanoma subtypes. It identified an intermediate category of melanocytic neoplasia, characterized by the presence of more than one pathogenic genetic alteration and distinctive histopathological features. Finally, our study implicated ultraviolet radiation as a major factor in both the initiation and progression of melanoma. (Funded by the National Institutes of Health and others.).


Assuntos
Evolução Molecular , Melanoma/genética , Mutação , Nevo Pigmentado/genética , Neoplasias Cutâneas/genética , Raios Ultravioleta/efeitos adversos , Variações do Número de Cópias de DNA , Progressão da Doença , Humanos , Melanoma/patologia , Nevo Pigmentado/patologia , Mutação Puntual , Análise de Sequência de DNA , Neoplasias Cutâneas/patologia
3.
Mod Pathol ; 31(4): 660-673, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29148537

RESUMO

Adenomatoid tumors are the most common neoplasm of the epididymis, and histologically similar adenomatoid tumors also commonly arise in the uterus and fallopian tube. To investigate the molecular pathogenesis of these tumors, we performed genomic profiling on a cohort of 31 adenomatoid tumors of the male and female genital tracts. We identified that all tumors harbored somatic missense mutations in the TRAF7 gene, which encodes an E3 ubiquitin ligase belonging to the family of tumor necrosis factor receptor-associated factors (TRAFs). These mutations all clustered into one of five recurrent hotspots within the WD40 repeat domains at the C-terminus of the protein. Functional studies in vitro revealed that expression of mutant but not wild-type TRAF7 led to increased phosphorylation of nuclear factor-kappa B (NF-kB) and increased expression of L1 cell adhesion molecule (L1CAM), a marker of NF-kB pathway activation. Immunohistochemistry demonstrated robust L1CAM expression in adenomatoid tumors that was absent in normal mesothelial cells, malignant peritoneal mesotheliomas and multilocular peritoneal inclusion cysts. Together, these studies demonstrate that adenomatoid tumors of the male and female genital tract are genetically defined by TRAF7 mutation that drives aberrant NF-kB pathway activation.


Assuntos
Tumor Adenomatoide/genética , Neoplasias dos Genitais Femininos/genética , Neoplasias dos Genitais Masculinos/genética , Peptídeos e Proteínas Associados a Receptores de Fatores de Necrose Tumoral/genética , Tumor Adenomatoide/metabolismo , Tumor Adenomatoide/patologia , Adulto , Idoso , Feminino , Neoplasias dos Genitais Femininos/metabolismo , Neoplasias dos Genitais Femininos/patologia , Neoplasias dos Genitais Masculinos/metabolismo , Neoplasias dos Genitais Masculinos/patologia , Humanos , Masculino , Pessoa de Meia-Idade , Mutação de Sentido Incorreto , NF-kappa B/metabolismo , Transdução de Sinais/fisiologia
4.
Mod Pathol ; 30(2): 246-254, 2017 02.
Artigo em Inglês | MEDLINE | ID: mdl-27813512

RESUMO

Malignant mesothelioma is a rare cancer that arises from the mesothelial cells that line the pleural cavity and less commonly from the peritoneal lining of the abdomen and pelvis. Most pleural mesotheliomas arise in patients with a history of asbestos exposure, whereas the association of peritoneal mesotheliomas with exposure to asbestos and other potential carcinogens is less clear, suggesting that the genetic alterations that drive malignant peritoneal mesothelioma may be unique from those in pleural mesothelioma. Treatment options for all malignant mesotheliomas are currently limited, with no known targeted therapies available. To better understand the molecular pathogenesis of malignant peritoneal mesothelioma, we sequenced 510 cancer-related genes in 13 patients with malignant mesothelioma arising in the peritoneal cavity. The most frequent genetic alteration was biallelic inactivation of the BAP1 gene, which occurred in 9/13 cases, with an additional two cases demonstrating monoallelic loss of BAP1. All 11 of these cases demonstrated loss of BAP1 nuclear staining by immunohistochemistry, whereas two tumors without BAP1 alteration and all 42 cases of histologic mimics in peritoneum (8 multilocular peritoneal inclusion cyst, 6 well-differentiated papillary mesothelioma of the peritoneum, 16 adenomatoid tumor, and 12 low-grade serous carcinoma of the ovary) demonstrated intact BAP1 nuclear staining. Additional recurrently mutated genes in this cohort of malignant peritoneal mesotheliomas included NF2 (3/13), SETD2 (2/13), and DDX3X (2/13). While these genes are known to be recurrently mutated in pleural mesotheliomas, the frequencies are distinct in peritoneal mesotheliomas, with nearly 85% of peritoneal tumors harboring BAP1 alterations versus only 20-30% of pleural tumors. Together, these findings demonstrate the importance of epigenetic modifiers including BAP1, SETD2, and DDX3X in mesothelial tumorigenesis and suggest opportunities for targeted therapies.


Assuntos
RNA Helicases DEAD-box/genética , Epigênese Genética , Histona-Lisina N-Metiltransferase/genética , Neoplasias Pulmonares/genética , Mesotelioma/genética , Neoplasias Peritoneais/genética , Proteínas Supressoras de Tumor/genética , Ubiquitina Tiolesterase/genética , Idoso , Idoso de 80 Anos ou mais , Carcinogênese/genética , Carcinogênese/patologia , Feminino , Perfilação da Expressão Gênica , Humanos , Neoplasias Pulmonares/patologia , Masculino , Mesotelioma/patologia , Mesotelioma Maligno , Pessoa de Meia-Idade , Neoplasias Peritoneais/patologia , Adulto Jovem
5.
Mod Pathol ; 30(8): 1086-1099, 2017 08.
Artigo em Inglês | MEDLINE | ID: mdl-28548128

RESUMO

Secretory carcinomas of the breast are rare tumors with distinct histologic features, recurrent t(12;15)(p13;q25) translocation resulting in ETV6-NTRK3 gene fusion and indolent clinical behavior. Mammary analog secretory carcinomas arising in other sites are histopathologically similar to the breast tumors and also harbor ETV6-NTRK3 fusions. Breast secretory carcinomas are often triple (estrogen and progesterone receptor, HER2) negative with a basal-like immunophenotype. However, genomic studies are lacking, and whether these tumors share genetic features with other basal and/or triple negative breast cancers is unknown. Aside from shared ETV6-NTRK3 fusions, the genetic relatedness of secretory carcinomas arising in different sites is also uncertain. We immunoprofiled and sequenced 510 cancer-related genes in nine breast secretory carcinomas and six salivary gland mammary analog secretory carcinomas. Immunoprofiles of breast and salivary gland secretory carcinomas were similar. All the tumors showed strong diffuse MUC4 expression (n=15), and SOX10 was positive in all nine breast and in five out of six salivary gland tumors. All breast secretory carcinomas were triple negative or weakly ER-positive, and all tumors at both the sites expressed CK5/6 and/or EGFR, consistent with a basal-like phenotype. Sequencing revealed classic ETV6-NTRK3 fusion genes in all cases, including in carcinoma in situ of one breast tumor. Translocations were reciprocal and balanced in six out of nine breast and three out of six salivary gland tumors and were complex in three others. In contrast to most breast basal carcinomas, the mutational burden of secretory carcinomas was very low, and no additional pathogenic aberrations were identified in genes typically mutated in breast cancer. Five (56%) breast and two (33%) salivary gland tumors had simple genomes without copy number changes; the remainder had very few changes, averaging 1.3 per tumor. The ETV6-NTRK3 derivative chromosome was duplicated in one breast and one salivary gland tumor, and was the only copy number change in the latter. The findings highlight breast secretory carcinoma as a subtype more closely related to mammary analog secretory carcinoma than to basal/triple negative breast cancers of no special type. Lack of pathogenic mutations in common cancer-related genes suggests that ETV6-NTRK3 alone may suffice to drive these tumors and likely helps explain their indolent behavior.


Assuntos
Biomarcadores Tumorais/genética , Neoplasias da Mama/genética , Carcinoma Secretor Análogo ao Mamário/genética , Adolescente , Adulto , Idoso , Neoplasias da Mama/patologia , Feminino , Perfilação da Expressão Gênica , Humanos , Masculino , Carcinoma Secretor Análogo ao Mamário/patologia , Pessoa de Meia-Idade , Proteínas de Fusão Oncogênica/genética , Neoplasias das Glândulas Salivares/genética , Neoplasias das Glândulas Salivares/patologia , Adulto Jovem
6.
PLoS Comput Biol ; 12(4): e1004873, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-27100738

RESUMO

Germline copy number variants (CNVs) and somatic copy number alterations (SCNAs) are of significant importance in syndromic conditions and cancer. Massively parallel sequencing is increasingly used to infer copy number information from variations in the read depth in sequencing data. However, this approach has limitations in the case of targeted re-sequencing, which leaves gaps in coverage between the regions chosen for enrichment and introduces biases related to the efficiency of target capture and library preparation. We present a method for copy number detection, implemented in the software package CNVkit, that uses both the targeted reads and the nonspecifically captured off-target reads to infer copy number evenly across the genome. This combination achieves both exon-level resolution in targeted regions and sufficient resolution in the larger intronic and intergenic regions to identify copy number changes. In particular, we successfully inferred copy number at equivalent to 100-kilobase resolution genome-wide from a platform targeting as few as 293 genes. After normalizing read counts to a pooled reference, we evaluated and corrected for three sources of bias that explain most of the extraneous variability in the sequencing read depth: GC content, target footprint size and spacing, and repetitive sequences. We compared the performance of CNVkit to copy number changes identified by array comparative genomic hybridization. We packaged the components of CNVkit so that it is straightforward to use and provides visualizations, detailed reporting of significant features, and export options for integration into existing analysis pipelines. CNVkit is freely available from https://github.com/etal/cnvkit.


Assuntos
Variações do Número de Cópias de DNA , Software , Hibridização Genômica Comparativa/estatística & dados numéricos , Biologia Computacional , Genoma Humano , Estudo de Associação Genômica Ampla/estatística & dados numéricos , Sequenciamento de Nucleotídeos em Larga Escala/estatística & dados numéricos , Humanos , Hibridização in Situ Fluorescente/estatística & dados numéricos , Análise de Sequência de DNA/estatística & dados numéricos
7.
BMC Evol Biol ; 16: 7, 2016 Jan 06.
Artigo em Inglês | MEDLINE | ID: mdl-26738562

RESUMO

BACKGROUND: Many prokaryotic kinases that phosphorylate small molecule substrates, such as antibiotics, lipids and sugars, are evolutionarily related to Eukaryotic Protein Kinases (EPKs). These Eukaryotic-Like Kinases (ELKs) share the same overall structural fold as EPKs, but differ in their modes of regulation, substrate recognition and specificity-the sequence and structural determinants of which are poorly understood. RESULTS: To better understand the basis for ELK specificity, we applied a Bayesian classification procedure designed to identify sequence determinants responsible for functional divergence. This reveals that a large and diverse family of aminoglycoside kinases, characterized members of which are involved in antibiotic resistance, fall into major sub-groups based on differences in putative substrate recognition motifs. Aminoglycoside kinase substrate specificity follows simple rules of alternating hydroxyl and amino groups that is strongly correlated with variations at the DFG + 1 position. CONCLUSIONS: Substrate specificity determining features in small molecule kinases are mostly confined to the catalytic core and can be identified based on quantitative sequence and crystal structure comparisons.


Assuntos
Proteínas Quinases/classificação , Sequência de Aminoácidos , Teorema de Bayes , Proteínas Quinases/química , Proteínas Quinases/metabolismo , Estrutura Terciária de Proteína , Especificidade por Substrato
8.
Mod Pathol ; 29(9): 1012-27, 2016 09.
Artigo em Inglês | MEDLINE | ID: mdl-27255162

RESUMO

Malignant phyllodes tumors of the breast are poorly understood rare neoplasms with potential for aggressive behavior. Few efficacious treatment options exist for progressed or metastatic disease. The molecular features of malignant phyllodes tumors are poorly defined, and a deeper understanding of the genetics of these tumors may shed light on pathogenesis and progression and potentially identify novel treatment approaches. We sequenced 510 cancer-related genes in 10 malignant phyllodes tumors, including 5 tumors with liposarcomatous differentiation and 1 with myxoid chondrosarcoma-like differentiation. Intratumoral heterogeneity was assessed by sequencing two separate areas in 7 tumors, including non-heterologous and heterologous components of tumors with heterologous differentiation. Activating hotspot mutations in FGFR1 were identified in 2 tumors. Additional recurrently mutated genes included TERT promoter (6/10), TP53 (4/10), PIK3CA (3/10), MED12 (3/10), SETD2 (2/10) and KMT2D (2/10). Together, genomic aberrations in FGFR/EGFR PI-3 kinase and RAS pathways were identified in 8 (80%) tumors and included mutually exclusive and potentially actionable activating FGFR1, PIK3CA and BRAF V600E mutations, inactivating TSC2 mutation, EGFR amplification and PTEN loss. Seven (70%) malignant phyllodes tumors harbored TERT aberrations (six promoter mutations, one amplification). For comparison, TERT promoter mutations were identified by Sanger sequencing in 33% borderline (n=12) and no (0%, n=8) benign phyllodes tumors (P=0.391 and P=0.013 vs malignant tumors, respectively). Genetic features specific to liposarcoma, including CDK4/MDM2 amplification, were not identified. Copy number analysis revealed intratumoral heterogeneity and evidence for divergent tumor evolution in malignant phyllodes tumors with and without heterologous differentiation. Tumors with liposarcomatous differentiation revealed more chromosomal aberrations in non-heterologous components compared with liposarcomatous components. EGFR amplification was heterogeneous and present only in the non-heterologous component of one tumor with liposarcomatous differentiation. The results identify novel pathways involved in the pathogenesis of malignant phyllodes tumors, which significantly increase our understanding of tumor biology and have potential clinical impact.


Assuntos
Biomarcadores Tumorais/genética , Neoplasias da Mama/genética , Classe I de Fosfatidilinositol 3-Quinases/genética , Perfilação da Expressão Gênica/métodos , Genes ras , Tumor Filoide/genética , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/genética , Transdução de Sinais/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Neoplasias da Mama/enzimologia , Neoplasias da Mama/patologia , Diferenciação Celular , Análise Mutacional de DNA , Feminino , Predisposição Genética para Doença , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Imuno-Histoquímica , Pessoa de Meia-Idade , Mutação , Fenótipo , Tumor Filoide/enzimologia , Tumor Filoide/patologia , São Francisco , Transcriptoma , Adulto Jovem
9.
Hum Mutat ; 36(2): 175-86, 2015 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-25382819

RESUMO

Protein kinases represent a large and diverse family of evolutionarily related proteins that are abnormally regulated in human cancers. Although genome sequencing studies have revealed thousands of variants in protein kinases, translating "big" genomic data into biological knowledge remains a challenge. Here, we describe an ontological framework for integrating and conceptualizing diverse forms of information related to kinase activation and regulatory mechanisms in a machine readable, human understandable form. We demonstrate the utility of this framework in analyzing the cancer kinome, and in generating testable hypotheses for experimental studies. Through the iterative process of aggregate ontology querying, hypothesis generation and experimental validation, we identify a novel mutational hotspot in the αC-ß4 loop of the kinase domain and demonstrate the functional impact of the identified variants in epidermal growth factor receptor (EGFR) constitutive activity and inhibitor sensitivity. We provide a unified resource for the kinase and cancer community, ProKinO, housed at http://vulcan.cs.uga.edu/prokino.


Assuntos
Neoplasias/enzimologia , Proteínas Quinases/genética , Sequência de Aminoácidos , Animais , Antineoplásicos/farmacologia , Células CHO , Domínio Catalítico , Cricetinae , Cricetulus , Mineração de Dados , Gefitinibe , Ontologia Genética , Humanos , Interações Hidrofóbicas e Hidrofílicas , Bases de Conhecimento , Modelos Moleculares , Neoplasias/genética , Inibidores de Proteínas Quinases/farmacologia , Proteínas Quinases/química , Quinazolinas/farmacologia , Alinhamento de Sequência , Software
10.
PLoS Comput Biol ; 10(4): e1003545, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24743239

RESUMO

Cancer is a genetic disease that develops through a series of somatic mutations, a subset of which drive cancer progression. Although cancer genome sequencing studies are beginning to reveal the mutational patterns of genes in various cancers, identifying the small subset of "causative" mutations from the large subset of "non-causative" mutations, which accumulate as a consequence of the disease, is a challenge. In this article, we present an effective machine learning approach for identifying cancer-associated mutations in human protein kinases, a class of signaling proteins known to be frequently mutated in human cancers. We evaluate the performance of 11 well known supervised learners and show that a multiple-classifier approach, which combines the performances of individual learners, significantly improves the classification of known cancer-associated mutations. We introduce several novel features related specifically to structural and functional characteristics of protein kinases and find that the level of conservation of the mutated residue at specific evolutionary depths is an important predictor of oncogenic effect. We consolidate the novel features and the multiple-classifier approach to prioritize and experimentally test a set of rare unconfirmed mutations in the epidermal growth factor receptor tyrosine kinase (EGFR). Our studies identify T725M and L861R as rare cancer-associated mutations inasmuch as these mutations increase EGFR activity in the absence of the activating EGF ligand in cell-based assays.


Assuntos
Mutação , Neoplasias/enzimologia , Oncogenes , Proteínas Quinases/metabolismo , Inteligência Artificial , Humanos , Proteínas Quinases/genética
11.
J Proteome Res ; 12(9): 4028-45, 2013 Sep 06.
Artigo em Inglês | MEDLINE | ID: mdl-23914800

RESUMO

During asexual intraerythrocytic development, Plasmodium falciparum diverges from the paradigm of the eukaryotic cell cycles by undergoing multiple rounds of DNA replication and nuclear division without cytokinesis. A better understanding of the molecular switches that coordinate a myriad of events for the progression of the parasite through the intraerythrocytic developmental stages will be of fundamental importance for rational design of intervention strategies. To achieve this goal, we performed isobaric tag-based quantitative proteomics and phosphoproteomics analyses of three developmental stages in the Plasmodium asexual cycle and identified 2767 proteins, 1337 phosphoproteins, and 6293 phosphorylation sites. Approximately 34% of identified proteins and 75% of phosphorylation sites exhibit changes in abundance as the intraerythrocytic cycle progresses. Our study identified 43 distinct phosphorylation motifs and a range of potential MAPK/CDK substrates. Further analysis of phosphorylated kinases identified 30 protein kinases with 126 phosphorylation sites within the kinase domain or in N- or C-terminal tails. Many of these phosphorylations are likely CK2-mediated. We define the constitutive and regulated expression of the Plasmodium proteome during the intraerythrocytic developmental cycle, offering an insight into the dynamics of phosphorylation during asexual cycle progression. Our system-wide comprehensive analysis is a major step toward defining kinase-substrate pairs operative in various signaling networks in the parasite.


Assuntos
Eritrócitos/parasitologia , Plasmodium falciparum/metabolismo , Processamento de Proteína Pós-Traducional , Proteoma/metabolismo , Proteínas de Protozoários/metabolismo , Sequência de Aminoácidos , Células Cultivadas , Humanos , Dados de Sequência Molecular , Fosfoproteínas/química , Fosfoproteínas/isolamento & purificação , Fosfoproteínas/metabolismo , Fosforilação , Plasmodium falciparum/crescimento & desenvolvimento , Proteínas Quinases/química , Proteínas Quinases/isolamento & purificação , Proteínas Quinases/metabolismo , Proteoma/química , Proteoma/isolamento & purificação , Proteínas de Protozoários/química , Proteínas de Protozoários/isolamento & purificação , Transdução de Sinais , Coloração e Rotulagem
12.
BMC Evol Biol ; 13: 117, 2013 Jun 06.
Artigo em Inglês | MEDLINE | ID: mdl-23742205

RESUMO

BACKGROUND: The widespread protozoan parasite Toxoplasma gondii interferes with host cell functions by exporting the contents of a unique apical organelle, the rhoptry. Among the mix of secreted proteins are an expanded, lineage-specific family of protein kinases termed rhoptry kinases (ROPKs), several of which have been shown to be key virulence factors, including the pseudokinase ROP5. The extent and details of the diversification of this protein family are poorly understood. RESULTS: In this study, we comprehensively catalogued the ROPK family in the genomes of Toxoplasma gondii, Neospora caninum and Eimeria tenella, as well as portions of the unfinished genome of Sarcocystis neurona, and classified the identified genes into 42 distinct subfamilies. We systematically compared the rhoptry kinase protein sequences and structures to each other and to the broader superfamily of eukaryotic protein kinases to study the patterns of diversification and neofunctionalization in the ROPK family and its subfamilies. We identified three ROPK sub-clades of particular interest: those bearing a structurally conserved N-terminal extension to the kinase domain (NTE), an E. tenella-specific expansion, and a basal cluster including ROP35 and BPK1 that we term ROPKL. Structural analysis in light of the solved structures ROP2, ROP5, ROP8 and in comparison to typical eukaryotic protein kinases revealed ROPK-specific conservation patterns in two key regions of the kinase domain, surrounding a ROPK-conserved insert in the kinase hinge region and a disulfide bridge in the kinase substrate-binding lobe. We also examined conservation patterns specific to the NTE-bearing clade. We discuss the possible functional consequences of each. CONCLUSIONS: Our work sheds light on several important but previously unrecognized features shared among rhoptry kinases, as well as the essential differences between active and degenerate protein kinases. We identify the most distinctive ROPK-specific features conserved across both active kinases and pseudokinases, and discuss these in terms of sequence motifs, evolutionary context, structural impact and potential functional relevance. By characterizing the proteins that enable these parasites to invade the host cell and co-opt its signaling mechanisms, we provide guidance on potential therapeutic targets for the diseases caused by coccidian parasites.


Assuntos
Evolução Molecular , Proteínas Quinases/química , Proteínas Quinases/genética , Toxoplasma/enzimologia , Fatores de Virulência/química , Fatores de Virulência/genética , Sequência de Aminoácidos , Sequência Conservada , Modelos Moleculares , Família Multigênica , Filogenia , Proteínas Quinases/metabolismo , Estrutura Terciária de Proteína , Toxoplasma/classificação , Toxoplasma/genética , Fatores de Virulência/metabolismo
13.
BMC Bioinformatics ; 13: 209, 2012 Aug 21.
Artigo em Inglês | MEDLINE | ID: mdl-22909249

RESUMO

BACKGROUND: Ongoing innovation in phylogenetics and evolutionary biology has been accompanied by a proliferation of software tools, data formats, analytical techniques and web servers. This brings with it the challenge of integrating phylogenetic and other related biological data found in a wide variety of formats, and underlines the need for reusable software that can read, manipulate and transform this information into the various forms required to build computational pipelines. RESULTS: We built a Python software library for working with phylogenetic data that is tightly integrated with Biopython, a broad-ranging toolkit for computational biology. Our library, Bio.Phylo, is highly interoperable with existing libraries, tools and standards, and is capable of parsing common file formats for phylogenetic trees, performing basic transformations and manipulations, attaching rich annotations, and visualizing trees. We unified the modules for working with the standard file formats Newick, NEXUS and phyloXML behind a consistent and simple API, providing a common set of functionality independent of the data source. CONCLUSIONS: Bio.Phylo meets a growing need in bioinformatics for working with heterogeneous types of phylogenetic data. By supporting interoperability with multiple file formats and leveraging existing Biopython features, this library simplifies the construction of phylogenetic workflows. We also provide examples of the benefits of building a community around a shared open-source project. Bio.Phylo is included with Biopython, available through the Biopython website, http://biopython.org.


Assuntos
Filogenia , Software , Biologia Computacional/métodos
14.
BMC Evol Biol ; 11: 321, 2011 Nov 02.
Artigo em Inglês | MEDLINE | ID: mdl-22047078

RESUMO

BACKGROUND: The Apicomplexa constitute an evolutionarily divergent phylum of protozoan pathogens responsible for widespread parasitic diseases such as malaria and toxoplasmosis. Many cellular functions in these medically important organisms are controlled by protein kinases, which have emerged as promising drug targets for parasitic diseases. However, an incomplete understanding of how apicomplexan kinases structurally and mechanistically differ from their host counterparts has hindered drug development efforts to target parasite kinases. RESULTS: We used the wealth of sequence data recently made available for 15 apicomplexan species to identify the kinome of each species and quantify the evolutionary constraints imposed on each family of apicomplexan kinases. Our analysis revealed lineage-specific adaptations in selected families, namely cyclin-dependent kinase (CDK), calcium-dependent protein kinase (CDPK) and CLK/LAMMER, which have been identified as important in the pathogenesis of these organisms. Bayesian analysis of selective constraints imposed on these families identified the sequence and structural features that most distinguish apicomplexan protein kinases from their homologs in model organisms and other eukaryotes. In particular, in a subfamily of CDKs orthologous to Plasmodium falciparum crk-5, the activation loop contains a novel PTxC motif which is absent from all CDKs outside Apicomplexa. Our analysis also suggests a convergent mode of regulation in a subset of apicomplexan CDPKs and mammalian MAPKs involving a commonly conserved arginine in the αC helix. In all recognized apicomplexan CLKs, we find a set of co-conserved residues involved in substrate recognition and docking that are distinct from metazoan CLKs. CONCLUSIONS: We pinpoint key conserved residues that can be predicted to mediate functional differences from eukaryotic homologs in three identified kinase families. We discuss the structural, functional and evolutionary implications of these lineage-specific variations and propose specific hypotheses for experimental investigation. The apicomplexan-specific kinase features reported in this study can be used in the design of selective kinase inhibitors.


Assuntos
Apicomplexa/enzimologia , Evolução Molecular , Proteínas Quinases/química , Proteínas Quinases/genética , Sequência de Aminoácidos , Apicomplexa/química , Apicomplexa/genética , Quinases Ciclina-Dependentes/química , Quinases Ciclina-Dependentes/genética , Quinases Ciclina-Dependentes/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Ligação Proteica , Proteínas Quinases/metabolismo , Especificidade por Substrato
15.
Genome Med ; 13(1): 98, 2021 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-34074327

RESUMO

BACKGROUND: Metagenomic next-generation sequencing (mNGS) of body fluids is an emerging approach to identify occult pathogens in undiagnosed patients. We hypothesized that metagenomic testing can be simultaneously used to detect malignant neoplasms in addition to infectious pathogens. METHODS: From two independent studies (n = 205), we used human data generated from a metagenomic sequencing pipeline to simultaneously screen for malignancies by copy number variation (CNV) detection. In the first case-control study, we analyzed body fluid samples (n = 124) from patients with a clinical diagnosis of either malignancy (positive cases, n = 65) or infection (negative controls, n = 59). In a second verification cohort, we analyzed a series of consecutive cases (n = 81) sent to cytology for malignancy workup that included malignant positives (n = 32), negatives (n = 18), or cases with an unclear gold standard (n = 31). RESULTS: The overall CNV test sensitivity across all studies was 87% (55 of 63) in patients with malignancies confirmed by conventional cytology and/or flow cytometry testing and 68% (23 of 34) in patients who were ultimately diagnosed with cancer but negative by conventional testing. Specificity was 100% (95% CI 95-100%) with no false positives detected in 77 negative controls. In one example, a patient hospitalized with an unknown pulmonary illness had non-diagnostic lung biopsies, while CNVs implicating a malignancy were detectable from bronchoalveolar fluid. CONCLUSIONS: Metagenomic sequencing of body fluids can be used to identify undetected malignant neoplasms through copy number variation detection. This study illustrates the potential clinical utility of a single metagenomic test to uncover the cause of undiagnosed acute illnesses due to cancer or infection using the same specimen.


Assuntos
Líquidos Corporais , Biópsia Líquida/métodos , Metagenoma , Metagenômica/métodos , Neoplasias/diagnóstico , Neoplasias/etiologia , Líquidos Corporais/microbiologia , Estudos de Casos e Controles , Biologia Computacional/métodos , Análise Citogenética , Gerenciamento Clínico , Suscetibilidade a Doenças , Citometria de Fluxo , Histocitoquímica , Humanos , Hibridização in Situ Fluorescente , Biópsia Líquida/normas , Metagenômica/normas , Neoplasias/metabolismo , Reprodutibilidade dos Testes , Sensibilidade e Especificidade
16.
JAMA Neurol ; 78(11): 1355-1366, 2021 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-34515766

RESUMO

Importance: Cerebrospinal fluid (CSF) cytologic testing and flow cytometry are insensitive for diagnosing neoplasms of the central nervous system (CNS). Such clinical phenotypes can mimic infectious and autoimmune causes of meningoencephalitis. Objective: To ascertain whether CSF metagenomic next-generation sequencing (mNGS) can identify aneuploidy, a hallmark of malignant neoplasms, in difficult-to-diagnose cases of CNS malignant neoplasm. Design, Setting, and Participants: Two case-control studies were performed at the University of California, San Francisco (UCSF). The first study used CSF specimens collected at the UCSF Clinical Laboratories between July 1, 2017, and December 31, 2019, and evaluated test performance in specimens from patients with a CNS malignant neoplasm (positive controls) or without (negative controls). The results were compared with those from CSF cytologic testing and/or flow cytometry. The second study evaluated patients who were enrolled in an ongoing prospective study between April 1, 2014, and July 31, 2019, with presentations that were suggestive of neuroinflammatory disease but who were ultimately diagnosed with a CNS malignant neoplasm. Cases of individuals whose tumors could have been detected earlier without additional invasive testing are discussed. Main Outcomes and Measures: The primary outcome measures were the sensitivity and specificity of aneuploidy detection by CSF mNGS. Secondary subset analyses included a comparison of CSF and tumor tissue chromosomal abnormalities and the identification of neuroimaging characteristics that were associated with test performance. Results: Across both studies, 130 participants were included (median [interquartile range] age, 57.5 [43.3-68.0] years; 72 men [55.4%]). The test performance study used 125 residual laboratory CSF specimens from 47 patients with a CNS malignant neoplasm and 56 patients with other neurological diseases. The neuroinflammatory disease study enrolled 12 patients and 17 matched control participants. The sensitivity of the CSF mNGS assay was 75% (95% CI, 63%-85%), and the specificity was 100% (95% CI, 96%-100%). Aneuploidy was detected in 64% (95% CI, 41%-83%) of the patients in the test performance study with nondiagnostic cytologic testing and/or flow cytometry, and in 55% (95% CI, 23%-83%) of patients in the neuroinflammatory disease study who were ultimately diagnosed with a CNS malignant neoplasm. Of the patients in whom aneuploidy was detected, 38 (90.5%) had multiple copy number variations with tumor fractions ranging from 31% to 49%. Conclusions and Relevance: This case-control study showed that CSF mNGS, which has low specimen volume requirements, does not require the preservation of cell integrity, and was orginally developed to diagnose neurologic infections, can also detect genetic evidence of a CNS malignant neoplasm in patients in whom CSF cytologic testing and/or flow cytometry yielded negative results with a low risk of false-positive results.


Assuntos
Biomarcadores Tumorais/líquido cefalorraquidiano , Neoplasias do Sistema Nervoso Central/líquido cefalorraquidiano , Neoplasias do Sistema Nervoso Central/diagnóstico , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Adulto , Idoso , Estudos de Casos e Controles , Feminino , Humanos , Masculino , Metagenômica , Pessoa de Meia-Idade , Sensibilidade e Especificidade , Análise de Sequência de DNA/métodos
17.
Cell Rep ; 29(3): 573-588.e7, 2019 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-31618628

RESUMO

BRAF fusions are detected in numerous neoplasms, but their clinical management remains unresolved. We identified six melanoma lines harboring BRAF fusions representative of the clinical cases reported in the literature. Their unexpected heterogeneous responses to RAF and MEK inhibitors could be categorized upon specific features of the fusion kinases. Higher expression level correlated with resistance, and fusion partners containing a dimerization domain promoted paradoxical activation of the mitogen-activated protein kinase (MAPK) pathway and hyperproliferation in response to first- and second-generation RAF inhibitors. By contrast, next-generation αC-IN/DFG-OUT RAF inhibitors blunted paradoxical activation across all lines and had their therapeutic efficacy further increased in vitro and in vivo by combination with MEK inhibitors, opening perspectives in the clinical management of tumors harboring BRAF fusions.


Assuntos
Resistencia a Medicamentos Antineoplásicos/genética , Melanoma/patologia , Proteínas de Fusão Oncogênica/metabolismo , Proteínas Proto-Oncogênicas B-raf/genética , Animais , Dimerização , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Melanoma/genética , Camundongos , Camundongos Nus , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Proteínas de Fusão Oncogênica/antagonistas & inibidores , Proteínas de Fusão Oncogênica/genética , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Transdução de Sinais , Vemurafenib/farmacologia , Proteínas ras/genética , Proteínas ras/metabolismo
18.
Pigment Cell Melanoma Res ; 32(2): 269-279, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30156010

RESUMO

The deubiquitinating enzyme BAP1 is mutated in a hereditary cancer syndrome with a high risk of mesothelioma and melanocytic tumors. Here, we show that Bap1 deletion in melanocytes cooperates with the constitutively active, oncogenic form of BRAF (BRAFV600E ) and UV to cause melanoma in mice, albeit at very low frequency. In addition, Bap1-null melanoma cells derived from mouse tumors are more aggressive and colonize and grow at distant sites more than their wild-type counterparts. Molecularly, Bap1-null melanoma cell lines have increased DNA damage measured by γH2aX and hyperubiquitination of histone H2a. Therapeutically, these Bap1-null tumors are completely responsive to BRAF- and MEK-targeted therapies. Therefore, BAP1 functions as a tumor suppressor and limits tumor progression in melanoma.


Assuntos
Carcinogênese/genética , Carcinogênese/patologia , Melanoma/genética , Melanoma/patologia , Mutação/genética , Proteínas Proto-Oncogênicas B-raf/genética , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/patologia , Proteínas Supressoras de Tumor/metabolismo , Ubiquitina Tiolesterase/metabolismo , Animais , Linhagem Celular Tumoral , Proliferação de Células , Dano ao DNA , Transição Epitelial-Mesenquimal/genética , Deleção de Genes , Regulação Neoplásica da Expressão Gênica , Histonas/metabolismo , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Melanócitos/metabolismo , Melanócitos/patologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Transcrição Gênica , Ubiquitinação , Melanoma Maligno Cutâneo
19.
Nat Commun ; 9(1): 810, 2018 02 23.
Artigo em Inglês | MEDLINE | ID: mdl-29476136

RESUMO

Chordoid glioma is a rare brain tumor thought to arise from specialized glial cells of the lamina terminalis along the anterior wall of the third ventricle. Despite being histologically low-grade, chordoid gliomas are often associated with poor outcome, as their stereotypic location in the third ventricle makes resection challenging and efficacious adjuvant therapies have not been developed. Here we performed genomic profiling on 13 chordoid gliomas and identified a recurrent D463H missense mutation in PRKCA in all tumors, which localizes in the kinase domain of the encoded protein kinase C alpha (PKCα). Expression of mutant PRKCA in immortalized human astrocytes led to increased phospho-ERK and anchorage-independent growth that could be blocked by MEK inhibition. These studies define PRKCA as a recurrently mutated oncogene in human cancer and identify a potential therapeutic vulnerability in this uncommon brain tumor.


Assuntos
Neoplasias do Ventrículo Cerebral/enzimologia , Glioma/enzimologia , Proteína Quinase C-alfa/química , Proteína Quinase C-alfa/genética , Terceiro Ventrículo/enzimologia , Adulto , Idoso , Neoplasias do Ventrículo Cerebral/genética , MAP Quinases Reguladas por Sinal Extracelular/genética , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Feminino , Glioma/genética , Humanos , Masculino , Pessoa de Meia-Idade , Mutação de Sentido Incorreto , Fosforilação , Domínios Proteicos , Proteína Quinase C-alfa/metabolismo
20.
Clin Cancer Res ; 23(20): 6070-6077, 2017 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-28751446

RESUMO

Purpose: Precise detection of copy number aberrations (CNA) from tumor biopsies is critically important to the treatment of metastatic prostate cancer. The use of targeted panel next-generation sequencing (NGS) is inexpensive, high throughput, and easily feasible, allowing single-nucleotide variant calls, but CNA estimation from this remains challenging.Experimental Design: We evaluated CNVkit for CNA identification from amplicon-based targeted NGS in a cohort of 110 fresh castration-resistant prostate cancer biopsies and used capture-based whole-exome sequencing (WES), array comparative genomic hybridization (aCGH), and FISH to explore the viability of this approach.Results: We showed that this method produced highly reproducible CNA results (r = 0.92), with the use of pooled germline DNA as a coverage reference supporting precise CNA estimation. CNA estimates from targeted NGS were comparable with WES (r = 0.86) and aCGH (r = 0.7); for key selected genes (BRCA2, MYC, PIK3CA, PTEN, and RB1), CNA estimation correlated well with WES (r = 0.91) and aCGH (r = 0.84) results. The frequency of CNAs in our population was comparable with that previously described (i.e., deep deletions: BRCA2 4.5%; RB1 8.2%; PTEN 15.5%; amplification: AR 45.5%; gain: MYC 31.8%). We also showed, utilizing FISH, that CNA estimation can be impacted by intratumor heterogeneity and demonstrated that tumor microdissection allows NGS to provide more precise CNA estimates.Conclusions: Targeted NGS and CNVkit-based analyses provide a robust, precise, high-throughput, and cost-effective method for CNA estimation for the delivery of more precise patient care. Clin Cancer Res; 23(20); 6070-7. ©2017 AACR.


Assuntos
Variações do Número de Cópias de DNA , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , Proteína BRCA2/genética , Biomarcadores Tumorais , Biópsia , Hibridização Genômica Comparativa , Biologia Computacional/métodos , Heterogeneidade Genética , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Masculino , Reprodutibilidade dos Testes , Sequenciamento do Exoma
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