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2.
Cancer Sci ; 111(2): 601-609, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31845427

RESUMO

Multiple hepatocellular carcinoma (HCC) is divided into two categories: intrahepatic metastasis (IM), which is a true relapse of HCC, and multicentric origin (MO), which is a second primary tumor. Clinical diagnosis of multiple HCC is usually made based on tumor location and/or time to recurrence; however, it is often difficult to distinguish the two types of multiple HCC. Using 41 matched pairs of multiple HCC specimens, we confirmed the accuracy of clinical diagnoses using exome sequence data and investigated the importance of discriminating the type of multiple HCC. Genomic analysis revealed that 18 (43.9%) patients diagnosed as having genomic IM had common mutations in a pair of HCC tumors with the main tumor of these patients being more progressive compared to those with genomic MO. The accuracy of clinical diagnosis based on lobe (Definition 1) and segment (Definition 2) were 68.3% and 78.0%, respectively. Intriguingly, recurrence ≥2 years after initial surgery for 3 patients was IM. The survival of patients with clinical IM was significantly shorter than for those with clinical MO based on both Definition 1 (P = 0.045) and Definition 2 (P = 0.043). However, mean survival was not different between the patients with genomic IM and those with MO (P = 0.364). Taken together, genomic analysis elucidated that liver cancer may spread more extensively and more slowly than previously thought. In addition, distinguishing multiple HCC as IM or MC may have provided biological information but was not of clinical importance with respect to patient prognosis.


Assuntos
Carcinoma Hepatocelular/genética , Neoplasias Hepáticas/genética , Metástase Neoplásica/genética , Sequenciamento Completo do Exoma/métodos , Adulto , Idoso , Idoso de 80 Anos ou mais , Carcinoma Hepatocelular/cirurgia , Células Clonais/metabolismo , Feminino , Hepatectomia , Humanos , Neoplasias Hepáticas/cirurgia , Masculino , Pessoa de Meia-Idade , Prognóstico
3.
Cancer Immunol Immunother ; 68(12): 1979-1993, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31686124

RESUMO

5T4 (trophoblast glycoprotein, TPBG) is a transmembrane tumor antigen expressed on more than 90% of primary renal cell carcinomas (RCC) and a wide range of human carcinomas but not on most somatic adult tissues. The favorable expression pattern has encouraged the development and clinical testing of 5T4-targeted antibody and vaccine therapies. 5T4 also represents a compelling and unexplored target for T-cell receptor (TCR)-engineered T-cell therapy. Our group has previously isolated high-avidity CD8+ T-cell clones specific for an HLA-A2-restricted 5T4 epitope (residues 17-25; 5T4p17). In this report, targeted single-cell RNA sequencing was performed on 5T4p17-specific T-cell clones to sequence the highly variable complementarity-determining region 3 (CDR3) of T-cell receptor α chain (TRA) and ß chain (TRB) genes. Full-length TRA and TRB sequences were cloned into lentiviral vectors and transduced into CD8+ T-cells from healthy donors. Redirected effector T-cell function against 5T4p17 was measured by cytotoxicity and cytokine release assays. Seven unique TRA-TRB pairs were identified. All seven TCRs exhibited high expression on CD8+ T-cells with transduction efficiencies from 59 to 89%. TCR-transduced CD8+ T-cells demonstrated redirected cytotoxicity and cytokine release in response to 5T4p17 on target-cells and killed 5T4+/HLA-A2+ kidney-, breast-, and colorectal-tumor cell lines as well as primary RCC tumor cells in vitro. TCR-transduced CD8+ T-cells also detected presentation of 5T4p17 in TAP1/2-deficient T2 target-cells. TCR-transduced T-cells redirected to recognize the 5T4p17 epitope from a broadly shared tumor antigen are of interest for future testing as a cellular immunotherapy strategy for HLA-A2+ subjects with 5T4+ tumors.


Assuntos
Linfócitos T CD8-Positivos/fisiologia , Vacinas Anticâncer/imunologia , Carcinoma de Células Renais/terapia , Epitopos de Linfócito T/metabolismo , Imunoterapia Adotiva/métodos , Neoplasias Renais/terapia , Glicoproteínas de Membrana/metabolismo , Linfócitos T CD8-Positivos/transplante , Carcinoma de Células Renais/imunologia , Células Clonais , Citotoxicidade Imunológica , Epitopos de Linfócito T/imunologia , Antígeno HLA-A2/metabolismo , Humanos , Neoplasias Renais/imunologia , Glicoproteínas de Membrana/imunologia , Receptores de Antígenos de Linfócitos T alfa-beta/genética
4.
BMC Bioinformatics ; 20(1): 555, 2019 Nov 08.
Artigo em Inglês | MEDLINE | ID: mdl-31703552

RESUMO

BACKGROUND: We previously introduced a random-effects model to analyze a set of patients, each of which has two distinct tumors. The goal is to estimate the proportion of patients for which one of the tumors is a metastasis of the other, i.e. where the tumors are clonally related. Matches of mutations within a tumor pair provide the evidence for clonal relatedness. In this article, using simulations, we compare two estimation approaches that we considered for our model: use of a constrained quasi-Newton algorithm to maximize the likelihood conditional on the random effect, and an Expectation-Maximization algorithm where we further condition the random-effect distribution on the data. RESULTS: In some specific settings, especially with sparse information, the estimation of the parameter of interest is at the boundary a non-negligible number of times using the first approach, while the EM algorithm gives more satisfactory estimates. This is of considerable importance for our application, since an estimate of either 0 or 1 for the proportion of cases that are clonal leads to individual probabilities being 0 or 1 in settings where the evidence is clearly not sufficient for such definitive probability estimates. CONCLUSIONS: The EM algorithm is a preferable approach for our clonality random-effect model. It is now the method implemented in our R package Clonality, making available an easy and fast way to estimate this model on a range of applications.


Assuntos
Algoritmos , Neoplasias/classificação , Probabilidade , Células Clonais , Simulação por Computador , Feminino , Humanos , Funções Verossimilhança
5.
Nature ; 574(7779): 538-542, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31645727

RESUMO

The most common causes of chronic liver disease are excess alcohol intake, viral hepatitis and non-alcoholic fatty liver disease, with the clinical spectrum ranging in severity from hepatic inflammation to cirrhosis, liver failure or hepatocellular carcinoma (HCC). The genome of HCC exhibits diverse mutational signatures, resulting in recurrent mutations across more than 30 cancer genes1-7. Stem cells from normal livers have a low mutational burden and limited diversity of signatures8, which suggests that the complexity of HCC arises during the progression to chronic liver disease and subsequent malignant transformation. Here, by sequencing whole genomes of 482 microdissections of 100-500 hepatocytes from 5 normal and 9 cirrhotic livers, we show that cirrhotic liver has a higher mutational burden than normal liver. Although rare in normal hepatocytes, structural variants, including chromothripsis, were prominent in cirrhosis. Driver mutations, such as point mutations and structural variants, affected 1-5% of clones. Clonal expansions of millimetres in diameter occurred in cirrhosis, with clones sequestered by the bands of fibrosis that surround regenerative nodules. Some mutational signatures were universal and equally active in both non-malignant hepatocytes and HCCs; some were substantially more active in HCCs than chronic liver disease; and others-arising from exogenous exposures-were present in a subset of patients. The activity of exogenous signatures between adjacent cirrhotic nodules varied by up to tenfold within each patient, as a result of clone-specific and microenvironmental forces. Synchronous HCCs exhibited the same mutational signatures as background cirrhotic liver, but with higher burden. Somatic mutations chronicle the exposures, toxicity, regeneration and clonal structure of liver tissue as it progresses from health to disease.


Assuntos
Células Clonais/citologia , Células Clonais/patologia , Fibrose/genética , Fibrose/patologia , Fígado/citologia , Fígado/metabolismo , Mutação , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Células Clonais/metabolismo , Análise Mutacional de DNA , Hepatócitos/citologia , Hepatócitos/metabolismo , Hepatócitos/patologia , Humanos , Fígado/patologia , Masculino , Pessoa de Meia-Idade , Filogenia , Células-Tronco/citologia , Células-Tronco/metabolismo , Células-Tronco/patologia
6.
Nature ; 574(7779): 532-537, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31645730

RESUMO

The colorectal adenoma-carcinoma sequence has provided a paradigmatic framework for understanding the successive somatic genetic changes and consequent clonal expansions that lead to cancer1. However, our understanding of the earliest phases of colorectal neoplastic changes-which may occur in morphologically normal tissue-is comparatively limited, as for most cancer types. Here we use whole-genome sequencing to analyse hundreds of normal crypts from 42 individuals. Signatures of multiple mutational processes were revealed; some of these were ubiquitous and continuous, whereas others were only found in some individuals, in some crypts or during certain periods of life. Probable driver mutations were present in around 1% of normal colorectal crypts in middle-aged individuals, indicating that adenomas and carcinomas are rare outcomes of a pervasive process of neoplastic change across morphologically normal colorectal epithelium. Colorectal cancers exhibit substantially increased mutational burdens relative to normal cells. Sequencing normal colorectal cells provides quantitative insights into the genomic and clonal evolution of cancer.


Assuntos
Colo/citologia , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Mutação , Sintomas Prodrômicos , Reto/citologia , Adenoma/genética , Adenoma/patologia , Idoso , Proteína Axina/genética , Carcinoma/genética , Carcinoma/patologia , Transformação Celular Neoplásica , Células Clonais/citologia , Células Clonais/metabolismo , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Variações do Número de Cópias de DNA , Análise Mutacional de DNA , Feminino , Humanos , Mucosa Intestinal/citologia , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patologia , Masculino , Pessoa de Meia-Idade , Células-Tronco/citologia , Células-Tronco/metabolismo
7.
Vet Microbiol ; 238: 108434, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31648728

RESUMO

Mycoplasma hyopneumoniae causes enzootic pneumonia (EP) in swine, a disease related to high economic losses in production systems. Epidemiological spread of M. hyopneumoniae clones was studied by multi-locus sequence typing (MLST) in several swine production regions but so far not in South America. Using MLST, we have therefore investigated M. hyopneumoniae clones circulating in farms from three main swine production regions in Brazil. Porcine lungs samples were collected between 2015 and 2016 in farms with EP outbreaks. Three geographically distant regions were selected, and 67 M. hyopneumoniae positive samples, each one from a different farm, were included in the study. The occurrence of five sequence types (ST) was demonstrated and the majority of the samples were identified as ST-69 (n = 60; 89.5%), followed by ST-70 (n = 3; 4.5%), ST-123 (n = 2; 3%), ST-124 (n = 1; 1.5%) and ST-127 (n = 1; 1.5%). There was no association of any specific ST with region or production system. The five STs were all new ones, probably representing unique Brazilian clones. ST-69 and ST-70 on one side and ST-123 and ST-124 on the other side are phylogenetically close, while ST-127 is singleton. In conclusion, our results showed a low variability and high clonality of M. hyopneumoniae genotypes from Brazilian farms affected by EP.


Assuntos
Mycoplasma hyopneumoniae/classificação , Mycoplasma hyopneumoniae/genética , Pneumonia Suína Micoplasmática/microbiologia , Animais , Brasil , Células Clonais , Fazendas , Variação Genética , Genótipo , Pulmão/microbiologia , Filogenia , Suínos/microbiologia
8.
Int J Nanomedicine ; 14: 7107-7121, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31564868

RESUMO

Background: Cervical cancer (CxCa) ranks as the fourth most prevalent women-related cancer worldwide. Therefore, there is a crucial need to develop newer treatment modalities. Ormeloxifene (ORM) is a non-steroidal, selective estrogen receptor modulator (SERM) that is used as an oral contraceptive in humans. Recent investigations suggest that ORM exhibits potent anti-cancer activity against various types of cancers. Nanoparticulates offer targeted delivery of anti-cancer drugs with minimal toxicity and promise newer approaches for cancer diagnosis and treatment. Therefore, the nanotherapy approach is superior compared to traditional chemotherapy, which is not site-specific and is often associated with various side effects. Methods: Pursuing this novel nanotherapy approach, our lab has recently developed ORM-loaded poly [lactic-co-glycolic acid] (PLGA), an FDA-approved biodegradable polymer, nanoparticles to achieve targeted drug delivery and improved bioavailability. Our optimized PLGA-ORM nanoformulation showed improved internalization in both dose- and energy-dependent manners, through endocytosis-mediated pathways in both Caski and SiHa cell lines. Additionally, we employed MTS and colony forming assays to determine the short- and long-term effects of PLGA-ORM on these cells. Results: Our results showed that this formulation demonstrated improved inhibition of cellular proliferation and clonogenic potential compared to free ORM. Furthermore, the PLGA-ORM nanoformulation exhibited superior anti-tumor activities in an orthotopic cervical cancer mouse model than free ORM. Conclusion: Collectively, our findings suggest that our novel nanoformulation has great potential for repurposing the drug and becoming a novel modality for CxCa management.


Assuntos
Benzopiranos/uso terapêutico , Nanopartículas/uso terapêutico , Neoplasias do Colo do Útero/tratamento farmacológico , Animais , Benzopiranos/farmacologia , Carcinogênese/efeitos dos fármacos , Carcinogênese/metabolismo , Carcinogênese/patologia , Linhagem Celular Tumoral , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Clonais , Modelos Animais de Doenças , Endocitose/efeitos dos fármacos , Eritrócitos/metabolismo , Feminino , Hemólise/efeitos dos fármacos , Humanos , Teste de Materiais , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Camundongos Nus , Copolímero de Ácido Poliláctico e Ácido Poliglicólico/química , Soro/química , Neoplasias do Colo do Útero/patologia
9.
Immunity ; 51(4): 606-608, 2019 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-31618653

RESUMO

Although immune checkpoint blockade (ICB) has yielded striking clinical responses in subsets of cancer patients, the mechanism of action is still unclear. In a recent issue of Nature Medicine, Yost et al., 2019 report that the T cell clones that dominate the intra-tumoral T cell landscape after ICB are distinct from those prior to treatment, a phenomenon referred to by the authors as "clonal replacement."


Assuntos
Neoplasias , Receptor de Morte Celular Programada 1 , Células Clonais , Humanos , Linfócitos T
10.
Nature ; 574(7776): 122-126, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31554970

RESUMO

B cells are important in the pathogenesis of many, and perhaps all, immune-mediated diseases. Each B cell expresses a single B cell receptor (BCR)1, and the diverse range of BCRs expressed by the total B cell population of an individual is termed the 'BCR repertoire'. Our understanding of the BCR repertoire in the context of immune-mediated diseases is incomplete, and defining this could provide new insights into pathogenesis and therapy. Here, we compared the BCR repertoire in systemic lupus erythematosus, anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis, Crohn's disease, Behçet's disease, eosinophilic granulomatosis with polyangiitis, and immunoglobulin A (IgA) vasculitis by analysing BCR clonality, use of immunoglobulin heavy-chain variable region (IGHV) genes and-in particular-isotype use. An increase in clonality in systemic lupus erythematosus and Crohn's disease that was dominated by the IgA isotype, together with skewed use of the IGHV genes in these and other diseases, suggested a microbial contribution to pathogenesis. Different immunosuppressive treatments had specific and distinct effects on the repertoire; B cells that persisted after treatment with rituximab were predominately isotype-switched and clonally expanded, whereas the inverse was true for B cells that persisted after treatment with mycophenolate mofetil. Our comparative analysis of the BCR repertoire in immune-mediated disease reveals a complex B cell architecture, providing a platform for understanding pathological mechanisms and designing treatment strategies.


Assuntos
Doenças do Sistema Imunitário/imunologia , Isotipos de Imunoglobulinas/análise , Isotipos de Imunoglobulinas/imunologia , Receptores de Antígenos de Linfócitos B/análise , Receptores de Antígenos de Linfócitos B/imunologia , Adulto , Idoso , Células Clonais/citologia , Células Clonais/imunologia , Humanos , Imunoglobulina A/análise , Imunoglobulina A/imunologia , Switching de Imunoglobulina/imunologia , Imunoglobulina G/análise , Imunoglobulina G/imunologia , Pessoa de Meia-Idade , Adulto Jovem
11.
Genomics Proteomics Bioinformatics ; 17(3): 287-296, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31479759

RESUMO

T cells and T cell receptors (TCRs) play pivotal roles in adaptive immune responses against tumors. The development of next-generation sequencing technologies has enabled the analysis of the TCRß repertoire usage. Given the scarce investigations on the TCR repertoire in lung cancer tissues, in this study, we analyzed TCRß repertoires in lung cancer tissues and the matched distant non-tumor lung tissues (normal lung tissues) from 15 lung cancer patients. Based on our results, the general distribution of T cell clones was similar between cancer tissues and normal lung tissues; however, the proportion of highly expanded clones was significantly higher in normal lung tissues than in cancer tissues (0.021% ±â€¯0.002% vs. 0.016% ±â€¯0.001%, P = 0.0054, Wilcoxon signed rank test). In addition, a significantly higher TCR diversity was observed in cancer tissues than in normal lung tissues (431.37 ±â€¯305.96 vs. 166.20 ±â€¯101.58, P = 0.0075, Mann-Whitney U test). Moreover, younger patients had a significantly higher TCR diversity than older patients (640.7 ±â€¯295.3 vs. 291.8 ±â€¯233.6, P = 0.036, Mann-Whitney U test), and the higher TCR diversity in tumors was significantly associated with worse cancer outcomes. Thus, we provided a comprehensive comparison of the TCR repertoires between cancer tissues and matched normal lung tissues and demonstrated the presence of distinct T cell immune microenvironments in lung cancer patients.


Assuntos
Neoplasias Pulmonares/imunologia , Receptores de Antígenos de Linfócitos T/metabolismo , Adulto , Idoso , Células Clonais , Feminino , Variação Genética , Humanos , Neoplasias Pulmonares/genética , Masculino , Pessoa de Meia-Idade , Receptores de Antígenos de Linfócitos T/genética , Microambiente Tumoral
12.
Ann Clin Lab Sci ; 49(4): 468-473, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31471335

RESUMO

OBJECTIVE: To explore the clonal origin pattern of cardiac myxoma and its relationship with recurrence of the disease. METHODS: 20 female patients diagnosed with cardiac myxoma underwent appropriate surgery and were followed-up after the treatment. The DNA of tumor tissues and pairing normal tissues from 20 patients were taken, with polymerase chain reaction (PCR) assay being used to amplify the HUMARA gene on X-chromosome, which could hint the tumor cloning state. Cases were divided into a polyclonal origin group and monoclonal origin group, according to the PCR result. The recurrence rate in the two groups was compared using Fisher's exact probability method. RESULTS: All tumors were successfully removed. PCR assay showed that the hybrid rate in tumors was 90.0% (18/20). Among them, 88.9%(16/18) of cases were identified as polyclonal origin and 11.1%(2/18) were identified as monoclonal origin. After 4 years of follow-up, the recurrence rate was 12.5(2/16) in the polyclonal origin group and 0%(0/2) in monoclonal origin group, with significant difference between the two groups (P<0.05). CONCLUSION: Cardiac myxoma is mostly of polyclonal original, and its polyclonal origin characteristics may contribute to tumor recurrence.


Assuntos
Células Clonais/patologia , Neoplasias Cardíacas/patologia , Mixoma/patologia , Recidiva Local de Neoplasia/patologia , Feminino , Neoplasias Cardíacas/genética , Heterozigoto , Humanos , Mixoma/genética , Receptores Androgênicos/genética
13.
Vet Immunol Immunopathol ; 215: 109903, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31420067

RESUMO

Sensitivity of clonality analysis based on immunoglobulin heavy chain (IGH) in canine cutaneous plasmacytoma is lower than that in diffuse large B cell lymphoma (DLBCL) because of somatic hypermutation occurring at the IGH locus. Therefore, this study aimed to improve the sensitivity of clonality analysis for canine cutaneous plasmacytoma. To achieve this, clonality analysis based on the immunoglobulin kappa chain (IGK) locus was established. Sensitivity and specificity were examined in genomic DNA extracted from formalin-fixed paraffin-embedded sections of cutaneous plasmacytomas, DLBCLs, and lymph nodes without lymphoma. Forward primers were designed based on the IGKV genes, and reverse primers were designed based on the IGKJ genes and kappa deleting element (Kde). Analysis using IGKV and IGKJ primers demonstrated clonality in 24 of 29 cutaneous plasmacytomas (82.8%), while analysis with primers for IGKV and Kde showed clonality in 16 of 29 cases (55.2%). In DLBCL, the IGKV and IGKJ primer set yielded clonality in 18 of 23 cases (78.3%), and the IGKV and Kde primer set yielded 9 of 23 cases (39.1%). No clonal results were obtained from 23 lymph nodes without lymphoma. Sensitivity of the IGKV and IGKJ primer set was significantly higher than that of the IGH primers reported previously. Thus, clonality analysis based on the IGK locus can be utilized for canine B cell tumors. In conclusion, clonality testing based on IGH and IGK may be beneficial as an adjunct tool for diagnosis of canine B cell tumors including cutaneous plasmacytoma.


Assuntos
Doenças do Cão/imunologia , Cadeias kappa de Imunoglobulina/genética , Linfoma de Células B/veterinária , Plasmocitoma/veterinária , Neoplasias Cutâneas/veterinária , Animais , Células Clonais , DNA de Neoplasias , Doenças do Cão/genética , Cães , Genes de Imunoglobulinas , Região de Junção de Imunoglobulinas/genética , Região Variável de Imunoglobulina/genética , Linfonodos/imunologia , Linfoma de Células B/genética , Linfoma de Células B/imunologia , Plasmocitoma/genética , Plasmocitoma/imunologia , Plasmocitoma/patologia , Sensibilidade e Especificidade , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/imunologia
14.
Cancer Sci ; 110(10): 3375-3381, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31436356

RESUMO

Cell-free DNA (cfDNA) analysis to detect circulating tumor DNA has been focused on monitoring malignant lymphomas. However, clonal hematopoiesis of indeterminate potential (CHIP)-associated mutations can also be detected by cfDNA analysis. Our aim is to investigate the origin of mutations detected in cfDNA among B-cell lymphoma patients. MYD88/CD79B, DNMT3A, and TP53 were chosen as genes of interest, representing each of the following categories: lymphoma driver genes, CHIP-related genes, and genes shared between lymphoma and CHIP. Seventy-five B-cell lymphoma patients were included in this retrospective study. Serum cfDNAs at time of complete metabolic response (CMR) were sequenced for TP53 (N = 75) and DNMT3A (N = 49). MYD88 p.L265P and CD79B p.Y196C/H mutations were analyzed in diffuse large B-cell lymphoma (DLBCL) patients whose tumor samples were available (N = 29). Two and seven mutations in TP53 and DNMT3A, respectively, were detected in cfDNA at CMR. These mutations were detected in either bone marrow mononuclear cells (BMMC) or PBMC. Although four DNMT3A mutations were also detected in tumors, median variant allele frequencies in the tumors (<1.0%) were significantly lower than those in both BMMC (6.1%) and serum (5.2%) obtained before the therapy. Conversely, five MYD88 and three CD79B mutations detected in tumors were confirmed in cfDNA before therapy, but not in BMMC nor in cfDNA at CMR. Thus, all TP53 and DNMT3A mutations detected in cfDNA at remission seemed to originate from CHIP rather than from residual disease. Results of liquid biopsy should be carefully interpreted, especially in genes shared between lymphomas and CHIP.


Assuntos
Células Clonais/química , DNA (Citosina-5-)-Metiltransferases/genética , Hematopoese , Linfoma de Células B/genética , Mutação , Proteína Supressora de Tumor p53/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Células da Medula Óssea/química , Ácidos Nucleicos Livres/genética , Feminino , Frequência do Gene , Humanos , Leucócitos Mononucleares/química , Biópsia Líquida , Masculino , Pessoa de Meia-Idade , Monócitos/química , Indução de Remissão , Estudos Retrospectivos , Análise de Sequência de DNA/métodos
15.
Virchows Arch ; 475(6): 693-699, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31267202

RESUMO

The bone is a frequent localization for lung non-small cell cancer metastasis; decalcification is required to permit tissue section. Pre-analytical conditions can influence the detection of immunohistochemical markers. The aim of our work is to evaluate PD-L1 expression in samples with delayed fixation and in decalcified tissue with chelating agent or acid at different time. Tumor-expressing PD-L1 and placental tissue were fixed at different times or decalcified with an acid decalcifier or EDTA for different durations. For 22C3 antibody, when tissues were decalcified with DC3, there was a significant decrease in the percentage of tumor cells or placental villi stained which after 4 h (p = 0.035 at 4 h). When EDTA is used for 22C3 antibody, there was a slight decrease in the percentage of stained tumor cells or villi but although there was a trend (p = 0.058 at 20 h), this was never statistically significant. For E1L3N antibody, when tissues were decalcified either with DC3 or EDTA, there was no significant decrease for the proportion of stained tumor cells or placental villi, neither for staining intensity for the first 24 h. The proportion of placental villi and tumor stained or intensity of staining was not significantly lower for any sample after delayed fixation also at 24 h for both PD-L1 clones. Delayed fixation does not affect the proportion of stained cell and intensity with PD-L1 immunohistochemistry. Decalcification also performed with EDTA lower the proportion and intensity of stained cells with PD-L1 immunohistochemistry.


Assuntos
Antígeno B7-H1/metabolismo , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Fixação de Tecidos , Anticorpos Monoclonais/imunologia , Biomarcadores Tumorais/metabolismo , Células Clonais/patologia , Feminino , Humanos , Imuno-Histoquímica/métodos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Gravidez , Coloração e Rotulagem/métodos , Fatores de Tempo , Fixação de Tecidos/métodos
16.
Nat Commun ; 10(1): 2960, 2019 07 04.
Artigo em Inglês | MEDLINE | ID: mdl-31273196

RESUMO

Clone collections of modified strains ("libraries") are a major resource for systematic studies with the yeast Saccharomyces cerevisiae. Construction of such libraries is time-consuming, costly and confined to the genetic background of a specific yeast strain. To overcome these limitations, we present CRISPR-Cas12a (Cpf1)-assisted tag library engineering (CASTLING) for multiplexed strain construction. CASTLING uses microarray-synthesized oligonucleotide pools and in vitro recombineering to program the genomic insertion of long DNA constructs via homologous recombination. One simple transformation yields pooled libraries with >90% of correctly tagged clones. Up to several hundred genes can be tagged in a single step and, on a genomic scale, approximately half of all genes are tagged with only ~10-fold oversampling. We report several parameters that affect tagging success and provide a quantitative targeted next-generation sequencing method to analyze such pooled collections. Thus, CASTLING unlocks avenues for increasing throughput in functional genomics and cell biology research.


Assuntos
Sistemas CRISPR-Cas/genética , Técnicas Genéticas , Saccharomyces cerevisiae/genética , Células Clonais , Biblioteca Gênica , Engenharia Genética , Genoma Fúngico , Proteínas de Fluorescência Verde/metabolismo , Proteínas Nucleares/metabolismo
17.
Nature ; 571(7764): 205-210, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31270459

RESUMO

The mammalian brain contains neurogenic niches that comprise neural stem cells and other cell types. Neurogenic niches become less functional with age, but how they change during ageing remains unclear. Here we perform single-cell RNA sequencing of young and old neurogenic niches in mice. The analysis of 14,685 single-cell transcriptomes reveals a decrease in activated neural stem cells, changes in endothelial cells and microglia, and an infiltration of T cells in old neurogenic niches. T cells in old brains are clonally expanded and are generally distinct from those in old blood, which suggests that they may experience specific antigens. T cells in old brains also express interferon-γ, and the subset of neural stem cells that has a high interferon response shows decreased proliferation in vivo. We find that T cells can inhibit the proliferation of neural stem cells in co-cultures and in vivo, in part by secreting interferon-γ. Our study reveals an interaction between T cells and neural stem cells in old brains, opening potential avenues through which to counteract age-related decline in brain function.


Assuntos
Envelhecimento/fisiologia , Encéfalo/citologia , Movimento Celular , Células-Tronco Neurais/citologia , Neurogênese , Análise de Célula Única , Nicho de Células-Tronco/fisiologia , Linfócitos T/citologia , Animais , Sangue , Proliferação de Células , Células Clonais/citologia , Técnicas de Cocultura , Células Endoteliais/citologia , Interferon gama/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microglia/citologia , Análise de Sequência de RNA , Transdução de Sinais , Linfócitos T/metabolismo , Transcriptoma/genética
18.
Nature ; 571(7765): 355-360, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31270458

RESUMO

Defining the transcriptomic identity of malignant cells is challenging in the absence of surface markers that distinguish cancer clones from one another, or from admixed non-neoplastic cells. To address this challenge, here we developed Genotyping of Transcriptomes (GoT), a method to integrate genotyping with high-throughput droplet-based single-cell RNA sequencing. We apply GoT to profile 38,290 CD34+ cells from patients with CALR-mutated myeloproliferative neoplasms to study how somatic mutations corrupt the complex process of human haematopoiesis. High-resolution mapping of malignant versus normal haematopoietic progenitors revealed an increasing fitness advantage with myeloid differentiation of cells with mutated CALR. We identified the unfolded protein response as a predominant outcome of CALR mutations, with a considerable dependency on cell identity, as well as upregulation of the NF-κB pathway specifically in uncommitted stem cells. We further extended the GoT toolkit to genotype multiple targets and loci that are distant from transcript ends. Together, these findings reveal that the transcriptional output of somatic mutations in myeloproliferative neoplasms is dependent on the native cell identity.


Assuntos
Genótipo , Mutação , Transtornos Mieloproliferativos/genética , Transtornos Mieloproliferativos/patologia , Neoplasias/genética , Neoplasias/patologia , Transcriptoma/genética , Animais , Antígenos CD34/metabolismo , Calreticulina/genética , Linhagem Celular , Proliferação de Células , Células Clonais/classificação , Células Clonais/metabolismo , Células Clonais/patologia , Endorribonucleases/metabolismo , Hematopoese/genética , Células-Tronco Hematopoéticas/classificação , Células-Tronco Hematopoéticas/metabolismo , Células-Tronco Hematopoéticas/patologia , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Camundongos , Modelos Moleculares , Transtornos Mieloproliferativos/classificação , NF-kappa B/metabolismo , Neoplasias/classificação , Células-Tronco Neoplásicas/citologia , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Mielofibrose Primária/genética , Mielofibrose Primária/patologia , Proteínas Serina-Treonina Quinases/metabolismo , Análise de Sequência de RNA/métodos , Análise de Célula Única/métodos , Resposta a Proteínas não Dobradas/genética
19.
Hematology ; 24(1): 533-537, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31280705

RESUMO

OBJECTIVE: Buffy coat and ficoll of bone marrow (BM) are viable options for the study of minimal residual disease (MRD) in multiple myeloma (MM). As yet, there is no data about the superiority of either sample types. Herein, we aimed to address this issue. METHODS: Forty pairs of ficolled BMs and BM buffy coats of 19 MM patients were studied for MRD by allele-specific oligonucleotide real-time quantitative PCR, with patient-specific primers/probes whenever appropriate. RESULTS: There were 41 pairs of MRD data for comparison analysis due to one patient with biclonal disease. MRD levels in ficolls and buffy coats were highly concordant (rs = 0.936, P < 0.0001), with 31 (76%) and seven (17%) pairs being concomitantly MRD-positive or -negative. On the other hand, apart from the 16 pairs being both MRD-negative, or -positive but not quantifiable in ficolls and buffy coats, majority (n = 22, 88%) had higher MRD levels in ficolled BMs than BM buffy coats. Furthermore, in 17 pairs, in which MRD was quantifiable in both, MRD levels in ficolled BMs were 3.1 times those of BM buffy coats (median, 567/105 vs. 184/105, P = 0.001). CONCLUSION: Taken together, ficolled BM is more sensitive than BM buffy coat for MRD detection in MM, hence should be recommended.


Assuntos
Exame de Medula Óssea/métodos , Separação Celular/métodos , Centrifugação/métodos , Ficoll , Mieloma Múltiplo/patologia , Centrifugação com Gradiente de Concentração/métodos , Células Clonais , Primers do DNA , DNA de Neoplasias/análise , DNA de Neoplasias/isolamento & purificação , Rearranjo Gênico , Genes de Imunoglobulinas , Humanos , Leucócitos Mononucleares , Mieloma Múltiplo/genética , Neoplasia Residual , Células-Tronco Neoplásicas , Plasmócitos , Reação em Cadeia da Polimerase em Tempo Real , Sensibilidade e Especificidade , Manejo de Espécimes
20.
Mol Med Rep ; 20(2): 1575-1582, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31257493

RESUMO

Hepatocellular carcinoma (HCC) is the most common type of liver cancer, and exhibits a high mortality rate. Sirtuin (SIRT)6 is a member of the sirtuin family, which may be useful targets in the treatment of tumors. The present study aimed to explore the expression of SIRT6 in numerous HCC cell lines and investigate the role of SIRT6 in the proliferation and apoptosis of the HCC cells, and the underlying mechanisms. Overexpression and silencing of SIRT6 were performed by transfection of Huh­7 cells with synthetic overexpression and small interfering RNA (siRNA) plasmids. Cell proliferation was evaluated using a Cell Counting Kit­8 assay. The apoptosis rate was measured via flow cytometry. Cloning efficiency was assessed using plate clone formation assays. The expression of mRNAs and proteins were determined via reverse transcription­quantitative PCR and western blot analyses, respectively. SIRT6 was overexpressed in Hep3B, Huh­7, MHCC­97H, MHCC­97L, MHCC­LM6, MHCC­LM3, YY­8103 and SK­hep­1 cell lines, compared with MIHA and HL­7702 normal liver cell lines. Overexpression of SIRT6 increased the proliferation of Huh­7 cells, upregulated the expression of Bcl­2 and phosphorylation of extracellular­signal regulated protein kinase (ERK), and decreased the expression of cleaved­caspase­3 and Bcl­2­associated X protein (Bax) in Huh­7 cells. siRNA­mediated silencing of SIRT6 decreased the proliferation and increased the apoptosis of Huh­7 cells, downregulated the expression of Bcl­2 and phosphorylated­ERK, and promoted the expression of cleaved­caspase­3 and Bax. The proliferation of Huh­7 cells was decreased using the ERK1/2 inhibitor U0126. The results suggested that SIRT6 affected the proliferation and apoptosis of HCC cells via the regulation of the ERK1/2 pathway, altering the activation of the intrinsic apoptosis pathway. SIRT6 may be a potential target for the treatment of HCC; however, its role requires further investigation.


Assuntos
Apoptose/genética , Regulação Neoplásica da Expressão Gênica , Hepatócitos/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase 3 Ativada por Mitógeno/genética , Sirtuínas/genética , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Células Clonais , Hepatócitos/patologia , Humanos , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Transdução de Sinais , Sirtuínas/antagonistas & inibidores , Sirtuínas/metabolismo , Proteína X Associada a bcl-2/genética , Proteína X Associada a bcl-2/metabolismo
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