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1.
Recent Results Cancer Res ; 215: 77-88, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31605224

RESUMO

Circulating tumor cells (CTCs) provide valuable information about the molecular evolution of cancers, as they may initially respond and ultimately progress on therapy. As intact tumor cells isolated from the bloodstream, CTCs also enable assessment of heterogeneous subpopulations, and their analysis may include DNA, RNA, and protein biomarkers. New microfluidic cell isolation strategies greatly facilitate the challenge of enriching viable tumor cells from the billions of hematopoietic cells within a standard blood specimen. While counting and characterization of enriched CTCs have primarily relied on immunostaining for tumor cell-specific antigens, new RNA-based analytic platforms are providing new insight into the identity of CTCs and providing new tools for clinical applications. Single-cell RNA sequencing of CTCs reveals a high degree of heterogeneity among cancer cells from a single individual, while new digital RNA-based amplification platforms may now allow high-sensitivity and high-throughput quantitative scoring of CTCs for clinical applications. Here, we focus on transcriptomic analysis of CTCs and its relevance in understanding metastatic cancer progression and in developing diagnostic assays to monitor cancer.


Assuntos
Separação Celular/métodos , Neoplasias/genética , Neoplasias/patologia , Células Neoplásicas Circulantes , RNA Neoplásico/análise , Progressão da Doença , Humanos , Neoplasias/diagnóstico , Células Neoplásicas Circulantes/metabolismo , RNA Neoplásico/genética
2.
Int J Radiat Oncol Biol Phys ; 106(1): 174-181, 2020 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-31525407

RESUMO

PURPOSE: We aimed to study radiation-induced gene expression changes and to identify differences in gene expression between patients with and without response to radiation therapy (RT) for invasive breast cancer with the purpose of exploring whether a predictive signature could be developed. Such a signature could assist in optimizing individualized locoregional treatment. METHODS AND MATERIALS: RNA-seq using next-generation sequencing was performed on fresh frozen samples from pretreatment biopsies and post-RT surgery specimens from patients with low-risk breast cancer treated within the multicenter preoperative accelerated partial breast irradiation trial. Patients were treated with preoperative RT (10 × 4 Gy in 10 days or 5 × 6 Gy in 5 days) and a lumpectomy 6 weeks thereafter. The response of the tumor to RT was evaluated by pathologic assessment. To analyze the gene expression data, unsupervised and supervised clustering was performed. Gene expression profiles were compared between biopsies of responders and nonresponders and between samples before and after RT. RESULTS: Ninety-four samples from 77 patients were analyzed: 68 pretreatment biopsies and 26 post-RT surgery specimens. Six patients had a (near) complete pathologic response, 3 patients had a good response, 32 patients had a partial response, and 22 patients had no or very limited response. Comparing patients with and without response to RT, 25 genes were significantly differentially expressed and were not linked to a pathway. Comparison of samples before and after RT identified significant changes in gene expression. Genes involved in p53 signaling, TNFA1 signaling, apoptosis, epithelial mesenchymal transition, and inflammatory response were upregulated. Genes involved in mitotic spindle, G2M checkpoint, and E2F targets were downregulated. CONCLUSIONS: Radiation-induced gene expression changes mainly involved p53 signaling, cell cycle regulation, DNA repair, and inflammatory response. No clinically significant differences could be identified in gene expression between patients with and without response to RT.


Assuntos
Neoplasias da Mama/radioterapia , Carcinoma Ductal de Mama/radioterapia , Regulação Neoplásica da Expressão Gênica/efeitos da radiação , Terapia Neoadjuvante , RNA Neoplásico/análise , Transcriptoma , Biópsia , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Neoplasias da Mama/cirurgia , Carcinoma Ductal de Mama/genética , Carcinoma Ductal de Mama/patologia , Carcinoma Ductal de Mama/cirurgia , Ensaios Clínicos Fase II como Assunto/estatística & dados numéricos , Feminino , Secções Congeladas , Perfilação da Expressão Gênica/métodos , Humanos , Mastectomia , Estudos Multicêntricos como Assunto/estatística & dados numéricos , Medicina de Precisão , RNA Mensageiro/análise , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , RNA Neoplásico/biossíntese , RNA Neoplásico/genética , Tolerância a Radiação/genética , Análise de Sequência de RNA , Resultado do Tratamento
4.
Cancer Genomics Proteomics ; 16(3): 147-153, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31018945

RESUMO

BACKGROUND: Infiltrating lobular carcinoma (ILC) is the second most common histologicaI subtype of breast cancer, accounting for 10% of all cases. ILC has a characteristic genomic profile. ILC shows a high frequency of cadherin 1 (CDH1) mutations, along with loss of phosphatase and tensin homolog (PTEN), activation of alpha serine/threonine kinase (AKT), and mutations in T-box transcription factor (TBX3) and forkhead box protein A1 (FOXA1). We suspected that another gene, von Willebrand factor (VWF), might also be part of the profile, since coagulation tests reveal significant differences in patients with breast cancer. MATERIALS AND METHODS: For newly-diagnosed breast cancer, the association between VWF and histology in the GDC Breast Cancer dataset in The Cancer Genome Atlas (TCGA) was evaluated. The following were used to access and analyze the data: Genomic Data Commons Data Portal (https://portal.gdc.cancer.gov/); Xena browser (https://xenabrowser.net); cBioportal (http://cbioportal.org); Oncomine (https://oncomine.org); and Prediction Analysis of Microarray 50 (PAM50). RESULTS: Patients with ILC had higher VWF RNA expression than patients with infiltrating ductal carcinoma and other histology. The difference of expression was present to the same degree in both pre-menopausal and post-menopausal patients. Nine alterations in VWF and PTEN were significantly co-occurrent. Considering all histologies in 843 samples, Tukey's honest significant difference post hoc test showed that VWF RNA expression of the normal subtype was significantly greater than that of the other subtypes (p<0.001). CONCLUSION: Our finding of significantly higher VWF RNA expression in the PAM50 normal (non-basal-like) breast cancer subtype suggests that VWF protein measurement might complement or corroborate PAM50 results. VWF and PAM50 results both suggesting a low risk of recurrence might make the decision whether to give chemotherapy easier, especially if VWF protein were an independent predictor.


Assuntos
Neoplasias da Mama/genética , Carcinoma Ductal de Mama/genética , Carcinoma Lobular/genética , RNA Neoplásico/genética , Fator de von Willebrand/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/genética , Mama/metabolismo , Neoplasias da Mama/classificação , Neoplasias da Mama/patologia , Carcinoma Ductal de Mama/classificação , Carcinoma Ductal de Mama/patologia , Carcinoma Lobular/classificação , Carcinoma Lobular/patologia , Estudos de Casos e Controles , Feminino , Seguimentos , Perfilação da Expressão Gênica , Humanos , Pessoa de Meia-Idade , Prognóstico , RNA Neoplásico/análise , Curva ROC
5.
Int J Biol Markers ; 34(2): 101-107, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30862241

RESUMO

BACKGROUND: Prostate and bladder cancers continue to be the first and fourth most common cancers in men worldwide; thus there is an urgent need for more accurate biomarkers that can detect these types of cancer in a non-invasive way. Liquid biopsy is a new non-invasive tool for diagnosis and with a virtually unlimited supply urine is even more attractive resource since urinary exosomes have been discovered to contain RNAs that are hallmarks of cancer. It is challenging to assay those secreting lower amounts of molecules. METHODS: This review, based on articles identified through a PubMed/MEDLINE search, comprehensively summarizes state of the art approaches used in the discovery and validation of exosomal RNA biomarkers purified from the urine for lower urinary tract cancer. RESULTS: The combination of PCA3 and ERG has shown a relatively good improvement in diagnostic performance; examples of other potential biomarkers and the methods utilized in their discovery are also discussed in this review. CONCLUSIONS: Of these last markers, to date there are still few data to implement these for routine diagnosis.


Assuntos
Biomarcadores Tumorais/genética , Exossomos/genética , Neoplasias da Próstata/diagnóstico , RNA Neoplásico/genética , Neoplasias da Bexiga Urinária/diagnóstico , Biomarcadores Tumorais/análise , Humanos , Masculino , Neoplasias da Próstata/genética , Neoplasias da Próstata/urina , RNA Neoplásico/análise , Neoplasias da Bexiga Urinária/genética , Neoplasias da Bexiga Urinária/urina
6.
Gastroenterology ; 156(6): 1761-1774, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30768984

RESUMO

BACKGROUND & AIMS: Esophageal adenocarcinoma (EAC) is resistant to standard chemoradiation treatments, and few targeted therapies are available. We used large-scale tissue profiling and pharmacogenetic analyses to identify deregulated signaling pathways in EAC tissues that might be targeted to slow tumor growth or progression. METHODS: We collected 397 biopsy specimens from patients with EAC and nonmalignant Barrett's esophagus (BE), with or without dysplasia. We performed RNA-sequencing analyses and used systems biology approaches to identify pathways that are differentially activated in EAC vs nonmalignant dysplastic tissues; pathway activities were confirmed with immunohistochemistry and quantitative real-time polymerase chain reaction analyses of signaling components in patient tissue samples. Human EAC (FLO-1 and EsoAd1), dysplastic BE (CP-B, CP-C, CP-D), and nondysplastic BE (CP-A) cells were incubated with pharmacologic inhibitors or transfected with small interfering RNAs. We measured effects on proliferation, colony formation, migration, and/or growth of xenograft tumors in nude mice. RESULTS: Comparisons of EAC vs nondysplastic BE tissues showed hyperactivation of transforming growth factor-ß (TGFB) and/or Jun N-terminal kinase (JNK) signaling pathways in more than 80% of EAC samples. Immunohistochemical analyses showed increased nuclear localization of phosphorylated JUN and SMAD proteins in EAC tumor tissues compared with nonmalignant tissues. Genes regulated by the TGFB and JNK pathway were overexpressed specifically in EAC and dysplastic BE. Pharmacologic inhibition or knockdown of TGFB or JNK signaling components in EAC cells (FLO-1 or EsoAd1) significantly reduced cell proliferation, colony formation, cell migration, and/or growth of xenograft tumors in mice in a SMAD4-independent manner. Inhibition of the TGFB pathway in BE cell lines reduced the proliferation of dysplastic, but not nondysplastic, cells. CONCLUSIONS: In a transcriptome analysis of EAC and nondysplastic BE tissues, we found the TGFB and JNK signaling pathways to be hyperactivated in EACs and the genes regulated by these pathways to be overexpressed in EAC and dysplastic BE. Inhibiting these pathways in EAC cells reduces their proliferation, migration, and formation of xenograft tumors. Strategies to block the TGFB and JNK signaling pathways might be developed for treatment of EAC.


Assuntos
Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Neoplasias Esofágicas/genética , Neoplasias Esofágicas/metabolismo , Sistema de Sinalização das MAP Quinases/genética , RNA Neoplásico/análise , Fator de Crescimento Transformador beta/metabolismo , Animais , Esôfago de Barrett/genética , Esôfago de Barrett/metabolismo , Esôfago de Barrett/patologia , Benzamidas/farmacologia , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Dioxóis/farmacologia , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Camundongos , Transplante de Neoplasias , Análise de Sequência com Séries de Oligonucleotídeos , Testes Farmacogenômicos , Proteínas Proto-Oncogênicas c-jun/metabolismo , Pirazóis/farmacologia , Quinolinas/farmacologia , Receptores de Fatores de Crescimento Transformadores beta/antagonistas & inibidores , Proteínas Smad/genética , Proteínas Smad/metabolismo , Biologia de Sistemas , Transcriptoma , Fator de Crescimento Transformador beta/antagonistas & inibidores , Fator de Crescimento Transformador beta/genética , Ensaio Tumoral de Célula-Tronco
7.
Gastric Cancer ; 22(4): 873-880, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-30603913

RESUMO

BACKGROUND: The double-tracer method has been established for sentinel node (SN) mapping in gastric cancer surgery. However, there remain several unresolved issues that prevent its widespread use in clinical practice. In this study, we aimed to demonstrate the feasibility of single-tracer SN mapping in laparoscopic surgery for gastric cancer, using indocyanine green (ICG) fluorescence imaging with a one-step nucleic acid amplification (OSNA) assay intraoperatively. METHODS: Patients with clinical T1N0M0 gastric adenocarcinoma preoperatively were considered for inclusion if they had a single primary lesion 4 cm or less in maximal diameter. Immunohistochemical staining with the anti-cytokeratin 19 antibody was performed on preoperative biopsy specimens, and patients with faint positive reactions were excluded. Intraoperatively, single-tracer SN biopsy with ICG fluorescence imaging was performed, followed by laparoscopic gastrectomy with modified D1+ or D2 lymph node dissection. RESULTS: Twenty eligible patients underwent SN biopsy and laparoscopic gastrectomy. SNs were identified in 17 cases (85%), with a median number of three SNs per patient. The median times for SN mapping and OSNA assay were 19 and 35 min, respectively. OSNA assay detected one metastatic lymph node, but all other nodes were negative. No adverse effects were observed in relation to SN mapping. CONCLUSIONS: Single-tracer SN mapping by ICG fluorescence imaging with intraoperative diagnosis by OSNA assay is feasible and safe. SNs can be identified in most patients, without producing false-negative results. Further clinical trial to demonstrate the sensitivity is ongoing.


Assuntos
Adenocarcinoma/cirurgia , Gastrectomia/métodos , Verde de Indocianina , Laparoscopia/métodos , Técnicas de Amplificação de Ácido Nucleico/métodos , Linfonodo Sentinela/patologia , Neoplasias Gástricas/cirurgia , Adenocarcinoma/genética , Adenocarcinoma/secundário , Adulto , Idoso , Idoso de 80 Anos ou mais , Estudos de Viabilidade , Feminino , Fluorescência , Corantes Fluorescentes/química , Seguimentos , Humanos , Masculino , Pessoa de Meia-Idade , Prognóstico , Estudos Prospectivos , RNA Neoplásico/análise , Neoplasias Gástricas/genética , Neoplasias Gástricas/patologia
8.
Lung Cancer ; 127: 6-11, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30642553

RESUMO

INTRODUCTION: RNA isolation from tumor tissue is used for biomarker analyses and validation. Limited diagnostic material from small volume biopsies combined with an increasing demand for standard histologic, molecular characterization, and next generation sequencing applications often leads to limited material for research. We sought to evaluate small volume sampling of lung cancer tissue collected from a single needle pass during a diagnostic procedure and determine if it can provide RNA of acceptable quantity and quality. METHODS: We enrolled 140 patients with probable primary bronchogenic carcinoma and collected RNA from a dedicated FNA aspiration. Total RNA (ηg), RNA integrity number (RIN), and %Mass in base pairs were evaluated from each patient sample. A customized nanoString nCounter® 95-gene panel was used to profile the expression patterns of feature NSCLC genes. We compared gene expression patterns that distinguish lung adenocarcinoma (LUAD) and squamous cell carcinoma (LUSC) in our cohort with a corresponding Cancer Genome Atlas (TCGA) NSCLC datasets. RESULTS: Of the 149 patients consented. RNA-extraction was performed in 101 eligible patients. A satisfactory total RNA mass and RIN was quantified for all samples with a similar distribution among cellular subtypes. Mean %-Mass over 300 base pairs was noted for all specimens and 96% of samples met criteria to perform genetic evaluation with our commercialized gene expression assay. The FNA-derived transcriptomic results showed excellent consistency with the TCGA counterparts, and the differential expression pattern of LUAD vs LUSC subtypes were highly similar. DISCUSSION: In this study, RNA retrieval from a single-pass FNA regardless of procedural approach showed equivalence and suitability for gene expression assessments. RNA extraction from small volume samples has the potential to provide valuable material for genetic profiling.


Assuntos
Biomarcadores Tumorais/análise , Biópsia por Agulha Fina/métodos , Carcinoma Broncogênico/diagnóstico , Neoplasias Pulmonares/diagnóstico , RNA Neoplásico/análise , Adulto , Idoso , Idoso de 80 Anos ou mais , Estudos de Coortes , Estudos de Viabilidade , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Pessoa de Meia-Idade , Transcriptoma
9.
Mikrochim Acta ; 186(2): 65, 2019 01 09.
Artigo em Inglês | MEDLINE | ID: mdl-30627866

RESUMO

A surface-enhanced Raman scattering (SERS) method is described for the determination of microRNA that is associated with various forms of cancer. The substrate consists of functionalized gold-silver bimetallic structure, and the sensitivity is strongly enhanced by making use of a re-circulated enzymatic amplification system (REAS). Poly-dopamine acts as both a reductant and a protective of the substrates. It was employed to link the gold core and silver satellite. The unique "hot spots" consisting of a Au@PDA@Ag nanocomposite improve the Raman signal and sensitivity. The reductive feature of PDA can prevent the susceptible oxidation of metallic silver to maintain the high Raman activity. To improve the sensitivity of the assays, a re-circulated enzymatic amplification system was developed in which the nicking endonuclease triggers the nucleic acid reaction system to enter an amplified cycle. By integrating the bimetallic nanosubstrate and magnetic separation into the REAS, microRNA can be detected by SERS (best at the Raman band of 1586 cm-1) with a limit of detection as low as 0.2 fM. In our perception, the assay provides an exciting new avenue to study the expression of tumor genes. Thus, it holds vast promise in cancer diagnosis. Graphical abstract Schematic presentation of the SERS method based on poly-dopamine mediated bimetallic SERS substrate and re-circulated enzymatic amplification.


Assuntos
MicroRNAs/análise , Nanocompostos/química , Neoplasias/diagnóstico , Análise Espectral Raman/métodos , Animais , Ouro , Humanos , Indóis/química , Técnicas de Amplificação de Ácido Nucleico , Polímeros/química , RNA Neoplásico/análise , Prata , Especificidade por Substrato
10.
Gut ; 68(2): 289-300, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30377189

RESUMO

OBJECTIVE: Campylobacter jejuni produces a genotoxin, cytolethal distending toxin (CDT), which has DNAse activity and causes DNA double-strand breaks. Although C. jejuni infection has been shown to promote intestinal inflammation, the impact of this bacterium on carcinogenesis has never been examined. DESIGN: Germ-free (GF) ApcMin/+ mice, fed with 1% dextran sulfate sodium, were used to test tumorigenesis potential of CDT-producing C. jejuni. Cells and enteroids were exposed to bacterial lysates to determine DNA damage capacity via γH2AX immunofluorescence, comet assay and cell cycle assay. To examine the interplay of CDT-producing C. jejuni, gut microbiome and host in tumorigenesis, colonic RNA-sequencing and faecal 16S rDNA sequencing were performed. Rapamycin was administrated to investigate the prevention of CDT-producing C. jejuni-induced tumorigenesis. RESULTS: GF ApcMin/+ mice colonised with human clinical isolate C. jejuni81-176 developed significantly more and larger tumours when compared with uninfected mice. C. jejuni with a mutated cdtB subunit, mutcdtB, attenuated C. jejuni-induced tumorigenesis in vivo and decreased DNA damage response in cells and enteroids. C. jejuni infection induced expression of hundreds of colonic genes, with 22 genes dependent on the presence of cdtB. The C. jejuni-infected group had a significantly different microbial gene expression profile compared with the mutcdtB group as shown by metatranscriptomic data, and different microbial communities as measured by 16S rDNA sequencing. Finally, rapamycin could diminish the tumorigenic capability of C. jejuni. CONCLUSION: Human clinical isolate C. jejuni 81-176 promotes colorectal cancer and induces changes in microbial composition and transcriptomic responses, a process dependent on CDT production.


Assuntos
Toxinas Bacterianas/toxicidade , Campylobacter jejuni/genética , Campylobacter jejuni/patogenicidade , Carcinogênese , Neoplasias Colorretais/genética , Neoplasias Colorretais/microbiologia , Animais , Campylobacter jejuni/isolamento & purificação , Dano ao DNA , DNA de Neoplasias/análise , Fezes/microbiologia , Microbioma Gastrointestinal , Expressão Gênica , Humanos , Camundongos , RNA Neoplásico/análise , Sirolimo/farmacologia , Transcriptoma
11.
Laryngoscope ; 129(1): 154-161, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30247749

RESUMO

OBJECTIVES/HYPOTHESIS: Gene expression analyses of head and neck cancer have revealed four molecular subtypes: basal (BA), mesenchymal (MS), atypical (AT), and classical (CL). We evaluate whether gene expression subtypes in oral cavity squamous cell carcinoma (OCSCC) and laryngeal squamous cell carcinoma (LSCC) can be used to predict nodal metastasis and prognosticate survival. STUDY DESIGN: Retrospective cohort study and genomic analysis. METHODS: OCSCC and LSCC cases were identified from the The Cancer Genome Atlas (TCGA) head and neck cancer cohort. RNA-seq by expected maximization (RSEM) was used to quantify gene expression levels from TCGA RNA-seq data and to assign each case to one of four subtypes. Descriptive statistics were used to describe patient, disease, and treatment characteristics in each subtype. Cox regression and Kaplan-Meier analyses were used to determine associations with survival. RESULTS: OCSCC cases were comprised primarily of the MS and BA subtypes, whereas LSCC was comprised primarily of CL and AT subtypes. In OCSCC, the MS subtype was significantly associated with higher risk of nodal metastasis. In a subset analysis of clinically T1-2N0M0 OCSCC, we demonstrate that the MS subtype was predictive of occult nodal metastasis (relative risk = 3.38, 95% confidence interval [CI]: 1.08-10.69). In LSCC, the CL subtype was associated with significantly worse overall survival (hazard ratio = 4.32, 95% CI: 1.77-10.54, P = .001). CONCLUSIONS: Gene expression analysis reveals potential novel markers of nodal metastasis and survival in human papillomavirus-negative head and neck cancer. Future studies will continue to refine and validate these markers, with the goal of providing molecular risk assessments that guide therapy and improve patient outcomes. LEVEL OF EVIDENCE: 2b Laryngoscope, 129:154-161, 2019.


Assuntos
Biomarcadores Tumorais/análise , Carcinoma de Células Escamosas/genética , Expressão Gênica , Neoplasias Laríngeas/genética , Neoplasias Bucais/genética , Biomarcadores Tumorais/genética , Carcinoma de Células Escamosas/mortalidade , Regulação Neoplásica da Expressão Gênica , Humanos , Estimativa de Kaplan-Meier , Neoplasias Laríngeas/mortalidade , Metástase Linfática/genética , Neoplasias Bucais/mortalidade , Modelos de Riscos Proporcionais , RNA Neoplásico/análise , Estudos Retrospectivos , Análise de Sequência de RNA
12.
N Engl J Med ; 379(24): 2330-2341, 2018 12 13.
Artigo em Inglês | MEDLINE | ID: mdl-30380364

RESUMO

BACKGROUND: As consolidation therapy for acute myeloid leukemia (AML), allogeneic hematopoietic stem-cell transplantation provides a benefit in part by means of an immune-mediated graft-versus-leukemia effect. We hypothesized that the immune-mediated selective pressure imposed by allogeneic transplantation may cause distinct patterns of tumor evolution in relapsed disease. METHODS: We performed enhanced exome sequencing on paired samples obtained at initial presentation with AML and at relapse from 15 patients who had a relapse after hematopoietic stem-cell transplantation (with transplants from an HLA-matched sibling, HLA-matched unrelated donor, or HLA-mismatched unrelated donor) and from 20 patients who had a relapse after chemotherapy. We performed RNA sequencing and flow cytometry on a subgroup of these samples and on additional samples for validation. RESULTS: On exome sequencing, the spectrum of gained and lost mutations observed with relapse after transplantation was similar to the spectrum observed with relapse after chemotherapy. Specifically, relapse after transplantation was not associated with the acquisition of previously unknown AML-specific mutations or structural variations in immune-related genes. In contrast, RNA sequencing of samples obtained at relapse after transplantation revealed dysregulation of pathways involved in adaptive and innate immunity, including down-regulation of major histocompatibility complex (MHC) class II genes ( HLA-DPA1, HLA-DPB1, HLA-DQB1, and HLA-DRB1) to levels that were 3 to 12 times lower than the levels seen in paired samples obtained at presentation. Flow cytometry and immunohistochemical analysis confirmed decreased expression of MHC class II at relapse in 17 of 34 patients who had a relapse after transplantation. Evidence suggested that interferon-γ treatment could rapidly reverse this phenotype in AML blasts in vitro. CONCLUSIONS: AML relapse after transplantation was not associated with the acquisition of relapse-specific mutations in immune-related genes. However, it was associated with dysregulation of pathways that may influence immune function, including down-regulation of MHC class II genes, which are involved in antigen presentation. These epigenetic changes may be reversible with appropriate therapy. (Funded by the National Cancer Institute and others.).


Assuntos
Genes MHC da Classe II/fisiologia , Transplante de Células-Tronco Hematopoéticas , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/imunologia , Mutação , Adolescente , Adulto , Idoso , Regulação para Baixo , Epigênese Genética , Feminino , Citometria de Fluxo , Humanos , Imunidade/genética , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/patologia , Masculino , Pessoa de Meia-Idade , RNA Neoplásico/análise , Recidiva , Análise de Sequência de RNA , Linfócitos T/imunologia , Transplante Homólogo , Sequenciamento Completo do Exoma
13.
PLoS One ; 13(10): e0198980, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30303958

RESUMO

Gastric cancer is the third most common cause of death from cancer in the world and it remains difficult to cure in Western countries, primarily because most patients present with advanced disease. Currently, CEA, CA50 and CA72-4 are commonly used as tumor markers for gastric cancer by immunoassays. However, the drawback and conundrum of immunoassay are the unceasing problem in standardization of quality of antibodies and time/effort for the intensive production. Therefore, there is an urgent need for the development of a standardized assay to detect gastric cancer at the early stage. Aptamers are DNA or RNA oligonucleotides with structural domain which recognize ligands such as proteins with superior affinity and specificity when compared to antibodies. In this study, SELEX (Systematic Evolution of Ligands by Exponential enrichment) technique was adopted to screen a random 30mer RNA library for aptamers targeting CEA, CA50 and CA72-4 respectively. Combined with high-throughput sequencing, we identified 6 aptamers which specifically target for these three biomarkers of gastrointestinal cancer. Intriguingly, the predicted secondary structures of RNA aptamers from each antigen showed significant structural similarity, suggesting the structural recognition between the aptamers and the antigens. Moreover, we determined the dissociation constants of all the aptamers to their corresponding antigens by fluorescence spectroscopy, which further demonstrated high affinities between the aptamers and the antigens. In addition, immunostaining of gastric adenocarcinoma cell line AGS using CEA Aptamer probe showed positive fluorescent signal which proves the potential of the aptamer as a detection tool for gastric cancer. Furthermore, substantially decreased cell viability and growth were observed when human colorectal cell line LS-174T was transfected with each individual aptamers. Taking together, these novel RNA aptamers targeting gastrointestinal cancer biomarker CEA, CA50 and CA72-4 will aid further development and standardization of clinical diagnostic method with better sensitivity and specificity, and potentially future therapeutics development of gastric cancer.


Assuntos
Antígenos Glicosídicos Associados a Tumores/análise , Aptâmeros de Nucleotídeos/química , Biomarcadores Tumorais/análise , Antígeno Carcinoembrionário/análise , Neoplasias Gastrointestinais/diagnóstico , Adenocarcinoma , Linhagem Celular Tumoral , Sobrevivência Celular , DNA/análise , Neoplasias Gastrointestinais/genética , Biblioteca Gênica , Células HeLa , Humanos , Prognóstico , RNA Neoplásico/análise , Técnica de Seleção de Aptâmeros , Sensibilidade e Especificidade
14.
Adv Exp Med Biol ; 1087: 109-117, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30259361

RESUMO

Circular RNAs (circRNAs) are a novel family of non-coding endogenous RNAs discovered in all eukaryotic cells and generated through a particular mechanism of alternative splicing called "back-splicing". These molecules show multiple functions, by acting as modulators of gene and miRNA expression, and may have a role in several biological processes, such as cell proliferation and invasion with, tumour development and progression, and in several mechanisms underlying other diseases. Their presence has been shown to be abundant in several body fluids such as blood and saliva. Based on their biogenesis mechanism, circRNAs may be categorized into five classes: exonic circRNAs, intronic circRNAs, antisense circRNAs, sense overlapping circRNAs and intergenic circRNAs. Recently, the presence of circRNAs, in addition to that of miRNAs and long non-coding RNAs, has been detected also in small extracellular vesicles called exosomes. Investigating the presence and expression levels of serum exosomal circRNAs could allow us, in future, to discriminate cancer patients from healthy individuals, identifying new potential exosome-based cancer biomarkers.In this chapter, we briefly will describe the major features and functions of exosomal circRNAs, discussing their potential role as molecular biomarkers for diagnosis, prognosis and monitoring of complex diseases, including cancer.


Assuntos
Exossomos/genética , RNA/genética , Líquidos Corporais/química , Comunicação Celular/genética , Detecção Precoce de Câncer/métodos , Previsões , Regulação da Expressão Gênica/genética , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Neoplasias/diagnóstico , Neoplasias/genética , RNA/análise , RNA Longo não Codificante/análise , RNA Longo não Codificante/genética , RNA Neoplásico/análise , RNA Neoplásico/genética
15.
Adv Exp Med Biol ; 1087: 141-157, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30259364

RESUMO

In the eukaryotic transcriptome, the evolutionary conserved circular RNAs naturally occur from the family of noncoding RNAs. Circular RNAs possess a unique feature to interact with nucleic acids and ribonucleoproteins and are establishing themselves as an obligatory composition for the regulatory messages which are encoded by the genome. The back-splicing mechanism leads to the formation of circularized RNA, and because of this they become resistant to exonuclease-mediated degradation. The differential and aberrant expression of circular RNAs can be detected with the help of various profiling methods by using serum, plasma, and tissue samples. In this chapter, we have highlighted the role of circular RNAs as putative biomarker for the detection of various human diseases along with its profiling methods. Here we have discussed the differentially expressed circular RNAs in neurological disorders and infectious diseases along with cancer diseases. For instance, in case of pulmonary tuberculosis, hsa_circRNA_001937 was upregulated, while hsa_circRNA_102101 got downregulated; Hsa_circ_000178 was depicted to get upregulated in breast cancer which is associated with disease progression. Furthermore, it has been observed that circRNAs are abundantly present within the mammalian brain tissues. In epileptic condition, Circ-EFCAB2 was observed to get notably upregulated within patients. Taking the above conditions into consideration, circular RNAs have proven themselves as promising noninvasive biomarker for the detection of human diseases.


Assuntos
Biomarcadores/análise , RNA/análise , Diagnóstico Precoce , Feminino , Hepatite Viral Humana/diagnóstico , Hepatite Viral Humana/genética , Humanos , MicroRNAs/metabolismo , Doença de Moyamoya/diagnóstico , Doença de Moyamoya/genética , Neoplasias/diagnóstico , Neoplasias/genética , Doenças do Sistema Nervoso/diagnóstico , Doenças do Sistema Nervoso/genética , Pré-Eclâmpsia/diagnóstico , Pré-Eclâmpsia/genética , Gravidez , Prognóstico , RNA/metabolismo , RNA Longo não Codificante/análise , RNA Longo não Codificante/metabolismo , RNA Neoplásico/análise , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sequência de RNA , Tuberculose/diagnóstico , Tuberculose/genética
16.
Adv Exp Med Biol ; 1087: 171-187, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30259366

RESUMO

As a type of novel noncoding RNAs, circular RNAs (circRNAs) have attracted great interest due to its different characteristics from linear RNAs. They are abundantly and stably present in the transcriptome of eukaryotic cells, with development stage specificity and high conservatism. Because circRNAs are not easily degraded by exonuclease RNase R, they can exist more stably in body fluids than linear RNAs. Based on these unique conditions, circRNAs have great potential value as clinical diagnostic and prognostic markers. As the research deepens, more and more evidences suggest that circRNAs may be closely associated with many diseases, especially cancer. Numerous studies have demonstrated the abnormal expression of circRNAs in cancer, and they can regulate the occurrence and progression of cancer by targeting key genes. Abundant circRNAs in tissues and cells can be released into saliva and blood. It is undeniable that circRNAs are a class of promising future biomarkers for cancer diagnosis and prognosis. Here we summarize the researches on circRNAs and cancer over the past few years. We expect this summary to be a stepping stone to further exploration of possible circRNAs as cancer biomarkers.


Assuntos
Biomarcadores Tumorais/análise , Neoplasias/diagnóstico , RNA Neoplásico/análise , RNA/análise , Bases de Dados Genéticas , Detecção Precoce de Câncer , Feminino , Previsões , Humanos , Masculino , Neoplasias/química , Neoplasias/genética , RNA/genética , RNA Neoplásico/genética
17.
Ann Surg Oncol ; 25(12): 3764-3770, 2018 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-30225832

RESUMO

BACKGROUND: Tissues from tumor patients are important resources for promoting cancer research, and therefore many biobanks have been established to collect tumor tissues; however, the quality of tumor tissues after surgical resection has not been well documented. METHODS: A total of 896 cases of tissues from 12 types of tumors were chosen for this study. First, histopathological examination was conducted to evaluate the tumor cell content; second, microchip electrophoresis was used to determine the RNA integrity number (RIN) in 466 cases of tissues with a tumor cell content ≥ 75%; and, finally, a correlation test was used to analyze the effect of ischemia on RNA integrity in 384 cases of tissues with a recorded ischemia time. RESULTS: Tumor tissues from 12 different organs had different tumor cell contents and RNA integrity. The liver had the highest percentage (69.7%) of tissue samples with a tumor cell content ≥ 75%, and the highest percentage (96%) of samples with an RIN ≥ 7. RNA integrity was not correlated with limited ex vivo ischemia time (5-60 min) in any of the 12 types of tumors. In contrast, a significant correlation with in vivo ischemia time was observed in several types of tumors. CONCLUSIONS: Not every sample of excised tumor tissue has a sufficient amount of tumor cells and enough RNA integrity. In vivo ischemia has a more significant influence on RNA integrity, and tumor tissues have different tolerances to pre-analytical variables. Those conducting translational research should pay attention to pre-analytical variables when collecting and utilizing tumor tissues.


Assuntos
Isquemia/fisiopatologia , Neoplasias/genética , Neoplasias/patologia , RNA Neoplásico/análise , Manejo de Espécimes/métodos , Humanos , RNA Neoplásico/genética , Fatores de Tempo , Bancos de Tecidos
18.
Oral Oncol ; 85: 44-51, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-30220319

RESUMO

OBJECTIVES: Heterogeneity of head and neck squamous cell carcinomas (HNSCCs) results in unpredictable outcomes for patients with similar stages of cancer. Beyond the role of human papilloma virus (HPV), no validated molecular marker of HNSCCs has been established. Thus, clinically relevant molecular subtypes are needed to optimize HNSCC therapy. The purpose of this study was to identify subtypes of HNSCC that have distinct biological characteristics associated with clinical outcomes and to characterize genomic alterations that best reflect the biological and clinical characteristics of each subtype. MATERIALS AND METHODS: We analyzed gene expression profiling data from pan-SCC tissues including cervical SCC, esophageal SCC, lung SCC, and HNSCC (n = 1346) to assess the similarities and differences among SCCs and to identify molecular subtypes of HNSCC associated with prognosis. Subtype-specific gene expression signatures were identified and used to construct predictive models. The association of the subtypes with prognosis was validated in two independent cohorts of patients. RESULTS: Pan-SCC analysis identified three novel subtypes of HNSCC. Subtype 1 had the best prognosis and was similar to cervical SCC, whereas subtype 3 had the worst prognosis and was similar to lung SCC. Subtype 2 had a moderate prognosis. The 600-gene signature associated with the three subtypes significantly predicted prognosis in two independent validation cohorts. These three subtypes also were associated with potential benefit of immunotherapy. CONCLUSION: We identified three clinically relevant HNSCC molecular subtypes. Independent prospective studies to assess the clinical utility of the subtypes and associated gene signature are warranted.


Assuntos
Carcinoma de Células Escamosas/classificação , Neoplasias de Cabeça e Pescoço/classificação , Carcinoma de Células Escamosas de Cabeça e Pescoço/classificação , Idoso , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/mortalidade , Carcinoma de Células Escamosas/terapia , Análise por Conglomerados , Estudos de Coortes , Feminino , Dosagem de Genes , Perfilação da Expressão Gênica , Neoplasias de Cabeça e Pescoço/genética , Neoplasias de Cabeça e Pescoço/mortalidade , Neoplasias de Cabeça e Pescoço/terapia , Humanos , Imunoterapia , Estimativa de Kaplan-Meier , Masculino , Pessoa de Meia-Idade , Infecções por Papillomavirus/genética , Infecções por Papillomavirus/mortalidade , Prognóstico , RNA Mensageiro/análise , RNA Neoplásico/análise , Carcinoma de Células Escamosas de Cabeça e Pescoço/genética , Carcinoma de Células Escamosas de Cabeça e Pescoço/mortalidade , Carcinoma de Células Escamosas de Cabeça e Pescoço/terapia , Resultado do Tratamento
19.
Gastroenterology ; 155(6): 1999-2013.e3, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30165049

RESUMO

BACKGROUND & AIMS: Genomic studies have revealed subtypes of pancreatic ductal adenocarcinoma (PDA) based on their molecular features, but different studies have reported different classification systems. It is a challenge to obtain high-quality, freshly frozen tissue for clinical analysis and determination of PDA subtypes. We aimed to redefine subtypes of PDA using a large number of formalin-fixed and paraffin-embedded PDA samples, which are more amenable to routine clinical evaluation. METHODS: We collected PDA samples from 309 consecutive patients who underwent surgery from September 1996 through December 2010 at 4 academic hospitals in Europe; nontumor tissue samples were not included. Samples were formalin fixed and paraffin embedded. DNA and RNA were isolated; gene expression, targeted DNA sequencing, and immunohistochemical analyses were performed. We used independent component analysis to deconvolute normal, tumor, and microenvironment transcriptome patterns in samples. We devised classification systems from an unsupervised analysis using a consensus clustering approach of our data set after removing normal contamination components. We associated subtypes with overall survival and disease-free survival of patients using Cox proportional hazards regression with estimation of hazard ratios and 95% confidence interval. We used The Cancer Genome Consortium and International Cancer Genome Consortium PDA data sets as validation cohorts. RESULTS: We validated the previously reported basal-like and classical tumor-specific subtypes of PDAs. We identified features of the PDA, including microenvironment gene expression patterns, that allowed tumors to be categorized into 5 subtypes, called pure basal like, stroma activated, desmoplastic, pure classical, and immune classical. These PDA subtypes have features of cancer cells and immune cells that could be targeted by pharmacologic agents. Tumor subtypes were associated with patient outcomes, based on analysis of our data set and the International Cancer Genome Consortium and The Cancer Genome Consortium PDA data sets. We also observed an exocrine signal associated with acinar cell contamination (from pancreatic tissue). CONCLUSIONS: We identified a classification system based on gene expression analysis of formalin-fixed PDA samples. We identified 5 PDA subtypes, based on features of cancer cells and the tumor microenvironment. This system might be used to select therapies and predict patient outcomes. We found evidence that the previously reported exocrine-like (called ADEX) tumor subtype resulted from contamination with pancreatic acinar cells. ArrayExpress accession number: E-MTAB-6134.


Assuntos
Adenocarcinoma/classificação , Carcinoma Ductal Pancreático/classificação , Microambiente Tumoral/genética , Células Acinares/patologia , Adenocarcinoma/genética , Adenocarcinoma/mortalidade , Adenocarcinoma/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/mortalidade , Carcinoma Ductal Pancreático/patologia , DNA de Neoplasias/análise , Intervalo Livre de Doença , Feminino , Expressão Gênica , Humanos , Masculino , Pessoa de Meia-Idade , Pâncreas/citologia , Pâncreas/patologia , Pâncreas Exócrino/metabolismo , Pâncreas Exócrino/patologia , Intervalo Livre de Progressão , Modelos de Riscos Proporcionais , Estudos Prospectivos , RNA Neoplásico/análise , Análise de Regressão , Análise de Sequência de DNA , Transcriptoma/genética
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