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Human colorectal cancers (CRCs) contain both clonal and subclonal mutations. Clonal driver mutations are positively selected, present in most cells, and drive malignant progression. Subclonal mutations are randomly dispersed throughout the genome, providing a vast reservoir of mutant cells that can expand, repopulate the tumor, and result in the rapid emergence of resistance, as well as being a major contributor to tumor heterogeneity. Here, we apply duplex sequencing (DS) methodology to quantify subclonal mutations in CRC tumor with unprecedented depth (104) and accuracy (<10-7). We measured mutation frequencies in genes encoding replicative DNA polymerases and in genes frequently mutated in CRC, and found an unexpectedly high effective mutation rate, 7.1 × 10-7. The curve of subclonal mutation accumulation as a function of sequencing depth, using DNA obtained from 5 different tumors, is in accord with a neutral model of tumor evolution. We present a theoretical approach to model neutral evolution independent of the infinite-sites assumption (which states that a particular mutation arises only in one tumor cell at any given time). Our analysis indicates that the infinite-sites assumption is not applicable once the number of tumor cells exceeds the reciprocal of the mutation rate, a circumstance relevant to even the smallest clinically diagnosable tumor. Our methods allow accurate estimation of the total mutation burden in clinical cancers. Our results indicate that no DNA locus is wild type in every malignant cell within a tumor at the time of diagnosis (probability of all cells being wild type, 10-308).
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Esophageal adenocarcinoma is the eighth most common malignancy worldwide. The overall prognosis is poor, with 5-year survival ranges of approximately 15-25%, and 30-50% for patients who can be treated with curative intent. There has been a marked increase in incidence of esophageal adenocarcinoma over the last 30 years, with chronic and severe reflux, diet and obesity identified as principal factors fuelling this rise in the West. Esophageal adenocarcinoma is an exemplar model of an inflammation-associated cancer. The key molecular pathways driving tumor development and influencing tumor biology are the subject of considerable research efforts, and is the principal focus of this review. In addition, the diverse range of changes occurring in the local immune response, tissue microenvironment, metabolic profile, intracellular signaling mechanisms and microRNA signatures are discussed, as well as novel targeted therapies.
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Adenocarcinoma/fisiopatología , Neoplasias Esofágicas/fisiopatología , Inflamación/fisiopatología , Adenocarcinoma/genética , Metabolismo Energético/fisiología , Neoplasias Esofágicas/genética , Reflujo Gastroesofágico/complicaciones , Reflujo Gastroesofágico/fisiopatología , Regulación Neoplásica de la Expresión Génica/fisiología , Inestabilidad Genómica/fisiología , Humanos , Inflamación/genética , MicroARNs/fisiología , Obesidad/complicaciones , Obesidad/fisiopatologíaRESUMEN
INTRODUCTION: A large body of evidence has implicated the signal transducer and activator of transcription (STAT) family and particularly the ubiquitously expressed STAT3 protein in the pathogenesis of colorectal, hepatocellular, gastric and pancreatic carcinoma. DISCUSSION: Concomitantly, an increasing body of epidemiological evidence has linked obesity and its associated pro-inflammatory state with the development of gastrointestinal cancers. Visceral adipose tissue is no longer considered inert and is known to secrete a number of adipocytokines such as leptin, interleukin (IL)-6, IL-8, IL-1ß and tumour necrosis factor-alpha (TNF-α) into the surrounding environment. Interestingly, these adipocytokines are strongly linked with the Janus kinase (JAK)/STAT pathway of signal transduction and there is experimental evidence linking IL-1ß, IL-8 and TNF-α to JAK/STAT signaling in other tissues. The result is an up-regulation of a wide range of anti-apoptotic, pro-metastatic and pro-angiogenic genes and processes. This is particularly relevant for gastrointestinal malignancy as these factors have the potential to signal adjacent endothelial cells in a paracrine manner. CONCLUSION: This review examines the potential role of the STAT3 signaling pathway in the pathogenesis of obesity-related gastrointestinal malignancy and the potential therapeutic role of STAT3 blockade given its status as a signaling hub for a number of inflammatory adipocytokines.
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Antineoplásicos/uso terapéutico , Neoplasias Gastrointestinales/tratamiento farmacológico , Neoplasias Gastrointestinales/etiología , Terapia Molecular Dirigida , Obesidad/complicaciones , Factor de Transcripción STAT3/antagonistas & inhibidores , Transducción de Señal/efectos de los fármacos , Animales , Neoplasias Gastrointestinales/metabolismo , Humanos , Factor de Transcripción STAT3/fisiologíaRESUMEN
BACKGROUND: Breast cancer is the most common female cancer worldwide. The lifetime risk of a woman being diagnosed with breast cancer is approximately 12.5%. For women who carry the deleterious mutation in either of the BRCA genes, BRCA1 or BRCA2, the risk of developing breast or ovarian cancer is significantly increased. In recent years there has been increased penetrance of BRCA1 and BRCA2 associated breast cancer, prompting investigation into the role of modifiable risk factors in this group. Previous investigations into this topic have relied on participants recalling lifetime weight changes and subjective methods of recording physical activity. The influence of obesity-related biomarkers, which may explain the link between obesity, physical activity and breast cancer risk, has not been investigated prospectively in this group. This paper describes the design of a prospective cohort study investigating the role of predictive and modifiable risk factors for breast cancer in unaffected BRCA1 and BRCA2 gene mutation carriers. METHODS/DESIGN: Participants will be recruited from breast cancer family risk clinics and genetics clinics. Lifestyle risk factors that will be investigated will include body composition, metabolic syndrome and its components, physical activity and dietary intake. PBMC telomere length will be measured as a potential predictor of breast cancer occurrence. Measurements will be completed on entry to the study and repeated at two years and five years. Participants will also be followed annually by questionnaire to track changes in risk factor status and to record cancer occurrence. Data will be analysed using multiple regression models. The study has an accrual target of 352 participants. DISCUSSION: The results from this study will provide valuable information regarding the role of modifiable lifestyle risk factors for breast cancer in women with a deleterious mutation in the BRCA gene. Additionally, the study will attempt to identify potential blood biomarkers which may be predictive of breast cancer occurrence.
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Neoplasias de la Mama/diagnóstico , Neoplasias de la Mama/etiología , Protocolos Clínicos , Femenino , Genes BRCA1 , Genes BRCA2 , Humanos , Mutación , Pronóstico , Estudios Prospectivos , Carácter Cuantitativo Heredable , Factores de RiesgoRESUMEN
We demonstrated that dendritic cell (DC) inhibition by tumor-conditioned media in the presence or absence of bevacizumab correlates with colorectal cancer patient survival. Monitoring the influence of the tumor microenvironment on infiltrating immune cells may offer an avenue for the discovery of biomarkers to guide the use of bevacizumab.
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Tumors inhibit dendritic cell maturation and function in order to evade host immunity. We showed that conditioned media from tumor explant tissue, taken from metastatic colorectal cancer patients, significantly inhibits maturation of dendritic cells.
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Cancer-related inflammation is considered the 'seventh hallmark of cancer'; many studies show that tumours develop and progress within inflammatory diseases. This review focuses on Barrett's oesophagus, a common condition in which chronic inflammation and resulting alterations in the stroma can lead to carcinogenesis, specifically oesophageal adenocarcinoma. Changes that occur in the tissue microenvironment during development of this disease are discussed. Infiltration of immune cells facilitates tumour development through production of factors that promote carcinogenesis and by enabling tumours to evade the host immune response. Small molecules including cytokines, chemokines and growth factors play key roles in both inflammation and cancer by promoting proliferation, angiogenesis and carcinogenesis and by recruiting immune cells. The extracellular matrix is altered in inflammation, and provides structural support to developing tumours. Hypoxia is a common state in cancers and inflamed tissues which causes DNA damage and induces tumourigenic factors. Finally, tissue vasculature is a vital part of its microenvironment, supplying oxygen, nutrients and growth factors to rapidly dividing cells, and providing a mechanism for metastatic spread. The cells and molecules outlined here represent potential targets for treatment of this cancer, especially in its pre-cancerous, inflammatory stage.
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Adenocarcinoma/patología , Esófago de Barrett/patología , Transformación Celular Neoplásica/patología , Neoplasias Esofágicas/patología , Esofagitis Péptica/complicaciones , Adenocarcinoma/irrigación sanguínea , Adenocarcinoma/metabolismo , Esófago de Barrett/etiología , Esófago de Barrett/metabolismo , Transformación Celular Neoplásica/inmunología , Citocinas/metabolismo , Neoplasias Esofágicas/irrigación sanguínea , Neoplasias Esofágicas/metabolismo , Matriz Extracelular/metabolismo , Humanos , Hipoxia/complicaciones , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Neovascularización Patológica , Estrés Oxidativo , Lesiones Precancerosas/etiología , Lesiones Precancerosas/patología , Linfocitos T/fisiologíaRESUMEN
Development of bevacizumab has improved survival in colorectal cancer, however, currently there are no biomarkers that predict response to bevacizumab and it is unknown how it influences the immune system in colorectal cancer patients. Dendritic cells are important for the induction of an antitumor immune response; however tumors are capable of disabling dendritic cells and escaping immune surveillance. The aim of this study was to assess the numbers of CD11c+ cells infiltrating tumor tissue and to examine the effects of tumor conditioned media (TCM) and bevacizumab conditioned media (BCM) on dendritic cell maturation and correlate our findings with patient survival. colorectal cancer explant tissues were cultured with or without bevacizumab, to generate BCM and TCM, which were used to treat dendritic cells. CD80, CD86, CD83, CD54, HLA-DR, and CD1d expression was measured by flow cytometry. Interleukin (IL)-10 and IL-12p70 were measured by ELISA. The Cox proportional hazards model was used to associate survival with dendritic cell inhibition. TCM and BCM inhibited lipopolysaccharide (LPS)-induced dendritic cell maturation and IL-12p70 secretion (P < 0.0001), while increasing IL-10 secretion (P = 0.0033 and 0.0220, respectively). Inhibition of LPS-induced CD1d (P = 0.021, HR = 1.096) and CD83 (P = 0.017, HR = 1.083) by TCM and inhibition of CD1d (P = 0.017, HR = 1.067), CD83 (P = 0.032, HR = 1.035), and IL-12p70 (P = 0.037, HR = 1.036) by BCM was associated with poor survival in colorectal cancer patients. CD11c expression was elevated in tumor tissue compared with normal tissue (P < 0.001), but this did not correlate with survival. In conclusion, TCM and BCM inhibit dendritic cells, and this inhibition correlates with survival of colorectal cancer patients receiving bevacizumab.
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Neoplasias Colorrectales/inmunología , Neoplasias Colorrectales/mortalidad , Células Dendríticas/inmunología , Microambiente Tumoral/inmunología , Adulto , Anciano , Anciano de 80 o más Años , Inhibidores de la Angiogénesis/administración & dosificación , Inhibidores de la Angiogénesis/uso terapéutico , Anticuerpos Monoclonales Humanizados/administración & dosificación , Anticuerpos Monoclonales Humanizados/uso terapéutico , Antígenos CD , Antígenos CD1d , Antineoplásicos/administración & dosificación , Antineoplásicos/uso terapéutico , Bevacizumab , Antígeno CD11c , Neoplasias Colorrectales/tratamiento farmacológico , Medios de Cultivo Condicionados/farmacología , Células Dendríticas/efectos de los fármacos , Femenino , Humanos , Inmunoglobulinas , Interleucina-10/metabolismo , Interleucina-12/metabolismo , Masculino , Glicoproteínas de Membrana/antagonistas & inhibidores , Persona de Mediana Edad , Estadificación de Neoplasias , Antígeno CD83RESUMEN
Inflammatory mediators in the tumour microenvironment promote tumour growth, vascular development and enable evasion of anti-tumour immune responses, by disabling infiltrating dendritic cells. However, the constituents of the tumour microenvironment that directly influence dendritic cell maturation and function are not well characterised. Our aim was to identify tumour-associated inflammatory mediators which influence the function of dendritic cells. Tumour conditioned media obtained from cultured colorectal tumour explant tissue contained high levels of the chemokines CCL2, CXCL1, CXCL5 in addition to VEGF. Pre-treatment of monocyte derived dendritic cells with this tumour conditioned media inhibited the up-regulation of CD86, CD83, CD54 and HLA-DR in response to LPS, enhancing IL-10 while reducing IL-12p70 secretion. We examined if specific individual components of the tumour conditioned media (CCL2, CXCL1, CXCL5) could modulate dendritic cell maturation or cytokine secretion in response to LPS. VEGF was also assessed as it has a suppressive effect on dendritic cell maturation. Pre-treatment of immature dendritic cells with VEGF inhibited LPS induced upregulation of CD80 and CD54, while CXCL1 inhibited HLA-DR. Interestingly, treatment of dendritic cells with CCL2, CXCL1, CXCL5 or VEGF significantly suppressed their ability to secrete IL-12p70 in response to LPS. In addition, dendritic cells treated with a combination of CXCL1 and VEGF secreted less IL-12p70 in response to LPS compared to pre-treatment with either cytokine alone. In conclusion, tumour conditioned media strongly influences dendritic cell maturation and function.
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Diferenciación Celular/inmunología , Neoplasias Colorrectales/inmunología , Células Dendríticas/inmunología , Microambiente Tumoral/inmunología , Anciano , Antígenos CD/inmunología , Antígenos CD/metabolismo , Antígeno B7-2/inmunología , Antígeno B7-2/metabolismo , Diferenciación Celular/efectos de los fármacos , Células Cultivadas , Quimiocina CCL2/inmunología , Quimiocina CCL2/metabolismo , Quimiocina CCL2/farmacología , Quimiocina CXCL1/inmunología , Quimiocina CXCL1/metabolismo , Quimiocina CXCL1/farmacología , Quimiocina CXCL5/inmunología , Quimiocina CXCL5/metabolismo , Quimiocina CXCL5/farmacología , Técnicas de Cocultivo , Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/patología , Medios de Cultivo Condicionados/metabolismo , Medios de Cultivo Condicionados/farmacología , Células Dendríticas/metabolismo , Ensayo de Inmunoadsorción Enzimática , Femenino , Citometría de Flujo , Antígenos HLA-DR/inmunología , Antígenos HLA-DR/metabolismo , Humanos , Inmunoglobulinas/inmunología , Inmunoglobulinas/metabolismo , Molécula 1 de Adhesión Intercelular/inmunología , Molécula 1 de Adhesión Intercelular/metabolismo , Interferón gamma/inmunología , Interferón gamma/metabolismo , Lipopolisacáridos/inmunología , Lipopolisacáridos/farmacología , Masculino , Glicoproteínas de Membrana/inmunología , Glicoproteínas de Membrana/metabolismo , Técnicas de Cultivo de Tejidos , Microambiente Tumoral/efectos de los fármacos , Factor A de Crecimiento Endotelial Vascular/inmunología , Factor A de Crecimiento Endotelial Vascular/metabolismo , Factor A de Crecimiento Endotelial Vascular/farmacología , Antígeno CD83RESUMEN
INTRODUCTION: To examine the effects of tumour necrosis factor (TNF) blocking therapy on the levels of early mitochondrial genome alterations and oxidative stress. METHODS: Eighteen inflammatory arthritis patients underwent synovial tissue oxygen (tpO(2)) measurements and clinical assessment of disease activity (DAS28-CRP) at baseline (T0) and three months (T3) after starting biologic therapy. Synovial tissue lipid peroxidation (4-HNE), T and B cell specific markers and synovial vascular endothelial growth factor (VEGF) were quantified by immunohistochemistry. Synovial levels of random mitochondrial DNA (mtDNA) mutations were assessed using Random Mutation Capture (RMC) assay. RESULTS: 4-HNE levels pre/post anti TNF-α therapy were inversely correlated with in vivo tpO(2) (P < 0.008; r = -0.60). Biologic therapy responders showed a significantly reduced 4-HNE expression (P < 0.05). High 4-HNE expression correlated with high DAS28-CRP (P = 0.02; r = 0.53), tender joint count for 28 joints (TJC-28) (P = 0.03; r = 0.49), swollen joint count for 28 joints (SJC-28) (P = 0.03; r = 0.50) and visual analogue scale (VAS) (P = 0.04; r = 0.48). Strong positive association was found between the number of 4-HNE positive cells and CD4+ cells (P = 0.04; r = 0.60), CD8+ cells (P = 0.001; r = 0.70), CD20+ cells (P = 0.04; r = 0.68), CD68+ cells (P = 0.04; r = 0.47) and synovial VEGF expression (P = 0.01; r = 063). In patients whose in vivo tpO(2) levels improved post treatment, significant reduction in mtDNA mutations and DAS28-CRP was observed (P < 0.05). In contrast in those patients whose tpO2 levels remained the same or reduced at T3, no significant changes for mtDNA mutations and DAS28-CRP were found. CONCLUSIONS: High levels of synovial oxidative stress and mitochondrial mutation burden are strongly associated with low in vivo oxygen tension and synovial inflammation. Furthermore these significant mitochondrial genome alterations are rescued following successful anti TNF-α treatment.
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Artritis Reumatoide/tratamiento farmacológico , Productos Biológicos/uso terapéutico , Estrés Oxidativo/efectos de los fármacos , Membrana Sinovial/efectos de los fármacos , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Artritis Reumatoide/genética , Hipoxia de la Célula/efectos de los fármacos , ADN Mitocondrial , Humanos , Inmunohistoquímica , Mitocondrias/genética , Mutagénesis , Mutación , Oxígeno/análisis , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
OBJECTIVE: To examine the effect of tumor necrosis factor (TNF) blocking therapy on hypoxia in vivo, macroscopic and microscopic inflammation, and magnetic resonance imaging (MRI) results in patients with inflammatory arthritis. METHODS: Patients with inflammatory arthritis (n = 20) underwent full clinical assessment, arthroscopy, synovial biopsy, and MRI before and after initiation of biologic therapy. Macroscopic synovitis/vascularity was assessed with a visual analog scale, and tissue PO(2) (tPO(2) ) was measured at arthroscopy using a Licox probe. Cell-specific markers (CD4, CD8, CD68, CD20, and CD19) and blood vessel maturity were quantified by immunohistologic analysis and dual-immunofluorescence factor VIII/α-smooth muscle actin staining, respectively. Contiguous gadoteric acid-enhanced MRI of the target knee was used to assess synovial enhancement. RESULTS: Biologic therapy responders showed a significant increase of tPO(2) in vivo (P < 0.05). This response was associated with significant reductions in 28-joint Disease Activity Score using the C-reactive protein level (DAS28-CRP) (P = 0.012), macroscopic synovitis (P = 0.017), macroscopic vascularity (P = 0.05), CD4+ T cells (P < 0.041), and CD68+ macrophages (P < 0.011). Blood vessel numbers were also reduced in responders; however, this did not reach statistical significance. Strong inverse correlations were demonstrated between changes in tPo(2) levels and changes in DAS28-CRP (r = -0.53, P < 0.001), CD4 (r = -0.44, P < 0.026), CD68 (r = -0.46, P < 0.003), and macroscopic vascularity (r = -0.314, P = 0.049) after therapy. Furthermore, changes in inflammation as measured by MRI showed a strong inverse correlation with tPO(2) levels (r = -0.688, P < 0.002) and positive correlations with CRP levels (r = 0.707, P = 0.001), macroscopic synovitis (r = 0.457, P = 0.056), macroscopic vascularity (r = 0.528, P= 0.017), CD4 (r = 0.553, P < 0.032), and CD68 (r = 0.670, P < 0.002) after therapy. CONCLUSION: This is the first study to show that successful biologic therapy significantly improves in vivo synovial hypoxia. Changes are strongly associated with changes in macroscopic and microscopic measures of joint inflammation and MRI improvement. These data further strengthen the concept that hypoxia is an important event driving synovial inflammation.
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Antirreumáticos/uso terapéutico , Artritis/tratamiento farmacológico , Terapia Biológica , Hipoxia/tratamiento farmacológico , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Adulto , Anciano , Anciano de 80 o más Años , Antígenos CD/metabolismo , Antígenos de Diferenciación Mielomonocítica/metabolismo , Artritis/metabolismo , Artritis/fisiopatología , Biopsia , Proteína C-Reactiva/metabolismo , Linfocitos T CD4-Positivos/patología , Estudios de Cohortes , Humanos , Hipoxia/metabolismo , Hipoxia/fisiopatología , Macrófagos/metabolismo , Macrófagos/patología , Imagen por Resonancia Magnética , Persona de Mediana Edad , Neovascularización Patológica/fisiopatología , Índice de Severidad de la Enfermedad , Membrana Sinovial/efectos de los fármacos , Membrana Sinovial/metabolismo , Membrana Sinovial/patologíaRESUMEN
Approximately 10% of ulcerative colitis patients develop colorectal neoplasia. At present, identification of this subset is markedly limited and necessitates lifelong colonoscopic surveillance for the entire ulcerative colitis population. Better risk markers are needed to focus surveillance onto the patients who are most likely to benefit. Using array-based comparative genomic hybridization, we analyzed single, non-dysplastic biopsies from three patient groups: ulcerative colitis progressors (n=9) with cancer or high-grade dysplasia at a mean distance of 18 cm from the analyzed site; ulcerative colitis non-progressors (n=8) without dysplasia during long-term surveillance; and non-ulcerative colitis normal controls (n=2). Genomic DNA from fresh colonic epithelium purified from stroma was hybridized to 287 (low-density) and 4342 (higher-density) feature bacterial artificial chromosome arrays. Sample-to-reference fluorescence ratios were calculated for individual chromosomal targets and globally across the genome. The low-density arrays yielded pronounced genomic gains and losses in 3 of 9 (33%) ulcerative colitis progressors but in none of the 10 control patients. Identical DNA samples analyzed on the higher-density arrays, using a combination of global and individual high variance assessments, distinguished all nine progressors from all 10 controls. These data confirm that genomic alterations in ulcerative colitis progressors are widespread, even involving single non-dysplastic biopsies that are far distant from neoplasia. They therefore show promise toward eliminating full colonoscopic surveillance with extensive biopsy sampling in the majority of ulcerative colitis patients.
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Biomarcadores de Tumor/genética , Colitis Ulcerosa/genética , Colitis Ulcerosa/patología , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/patología , Adulto , Edad de Inicio , Biopsia , Niño , Preescolar , Cromosomas Artificiales Bacterianos , Colitis Ulcerosa/complicaciones , Hibridación Genómica Comparativa , Progresión de la Enfermedad , Humanos , Hibridación Fluorescente in Situ , Análisis de Secuencia por Matrices de Oligonucleótidos , Adulto JovenRESUMEN
OBJECTIVES: To assess levels of oxidative DNA damage (8-oxo-7,8-dihydro-2'-deoxyguanine; 8-oxo-dG) and lipid peroxidation (4-hydroxy-2-nonenal; 4-HNE) in serum, synovial fluid and tissue of patients with inflammatory arthritis in relation to in vivo hypoxia levels, disease activity and angiogenic markers. METHODS: Oxygen levels in synovial tissue were assessed using an oxygen/temperature probe. Nuclear and cytoplasmic 8-oxo-dG and 4-HNE levels were assessed in synovial tissue from 23 patients by immunohistochemistry. 8-Oxo-dG and 4-HNE levels in serum and synovial fluid were determined using 8-oxo-dG and hexanoyl-Lys (HEL) adduct ELISAs, respectively. Serum vascular endothelial growth factor (VEGF) and angiopoietin 2 (Ang2) levels were also measured by ELISA. RESULTS: The median oxygen tension in synovial tissue was profoundly hypoxic at 19.35 mm Hg (2.5%). Nuclear 8-oxo-dG levels were significantly higher than nuclear 4-HNE levels in the lining and sublining layers (all p<0.001). In contrast, cytoplasmic 4-HNE levels were higher than cytoplasmic 8-oxo-dG levels in both cell layers (all p<0.001). Reduced in vivo oxygen tension correlated with high lipid peroxidation in synovial fluid (p=0.027; r=0.54) and tissue (p=0.004; r=0.58). Serum VEGF levels were positively correlated with cytoplasmic 4-HNE expression (p=0.05; r=0.43) and intensity (p=0.006; r=0.59) in the lining layer. Serum Ang2 levels were positively correlated with nuclear 4-HNE expression and intensity in both cell layers (all p < or = 0.05). DAS28-C-reactive protein was correlated with nuclear 4-HNE expression in the sublining layer (p=0.02; r=0.48) and DAS28-erythrocyte sedimentation rate was correlated with nuclear 4-HNE expression in both cell layers (p < or = 0.03). CONCLUSIONS: Lipid peroxidation is associated with low oxygen tension in vivo, disease activity and angiogenic marker expression in inflammatory arthritis.
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Artritis/metabolismo , Estrés Oxidativo/fisiología , Membrana Sinovial/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Inductores de la Angiogénesis/metabolismo , Artritis/sangre , Artritis/genética , Artroscopía , Biomarcadores/metabolismo , Sedimentación Sanguínea , Hipoxia de la Célula/fisiología , Daño del ADN , Femenino , Humanos , Peroxidación de Lípido/fisiología , Masculino , Persona de Mediana Edad , Oxígeno/sangre , Líquido Sinovial/metabolismoRESUMEN
Topoisomerase IIalpha is a nuclear enzyme that regulates the tertiary structure of DNA. The influence of topoisomerase IIalpha gene (TOP2A) or protein alterations on disease progression and treatment response in colorectal cancer (CRC) is unknown. The study investigated the clinical relevance of topoisomerase IIalpha in CRC using in vivo and in vitro models. Differentially expressed genes in early and late-stage CRC were identified by array comparative genomic hybridization (CGH). Cellular location of gene amplifications was determined by fluorescence in situ hybridization (FISH). Topoisomerase IIalpha levels, proliferation index, and HER2 expression were examined in 228 colorectal tumors by immunohistochemistry. Overexpression of topoisomerase IIalpha in vitro was achieved by liposome-based transfection. Cell growth inhibition and apoptosis were quantified using the crystal violet assay and flow cytometry, respectively, in response to drug treatment. Amplification of TOP2A was identified in 3 (7.7%) tumors using array CGH and confirmed using FISH. At the protein level, topoisomerase IIalpha staining was observed in 157 (69%) tumors, and both staining and intensity levels were associated with an aggressive tumor phenotype (p values 0.04 and 0.005, respectively). Using logistic regression analysis, topoisomerase IIalpha remained significantly associated with advanced tumor stage when corrected for tumor proliferation (p=0.007) and differentiation (p=0.001). No association was identified between topoisomerase IIalpha and HER2. In vitro, overexpression of topoisomerase IIalpha was associated with resistance to irinotecan (p=0.001) and etoposide chemotherapy (p=0.03), an effect mediated by inhibition of apoptosis. Topoisomerase IIalpha overexpression is significantly associated with alterations in tumor behavior and response to drug treatment in CRC. Our results suggest that gene amplification may represent an important mechanism underlying these changes.
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Antígenos de Neoplasias/fisiología , Apoptosis , Neoplasias Colorrectales/enzimología , ADN-Topoisomerasas de Tipo II/fisiología , Proteínas de Unión al ADN/fisiología , Adulto , Anciano , Anciano de 80 o más Años , Antígenos de Neoplasias/análisis , Antígenos de Neoplasias/genética , Proliferación Celular , Inestabilidad Cromosómica , Neoplasias Colorrectales/tratamiento farmacológico , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/patología , ADN-Topoisomerasas de Tipo II/análisis , ADN-Topoisomerasas de Tipo II/genética , Proteínas de Unión al ADN/análisis , Proteínas de Unión al ADN/genética , Resistencia a Antineoplásicos , Femenino , Humanos , Masculino , Persona de Mediana Edad , Proteínas de Unión a Poli-ADP-Ribosa , Receptor ErbB-2/análisisRESUMEN
No adequate means exist to identify the minority of ulcerative colitis (UC) patients destined to undergo neoplastic progression. Recognition of this subset would advance UC cancer surveillance by focusing the available management options onto the highest risk patients. Three different assays of genomic alterations in nondysplastic UC biopsies show promise for distinguishing patients with neoplasia (UC progressors) from those without (UC nonprogressors), including assays of telomere length, anaphase bridges, and chromosomal fluorescence in situ hybridization. Expanding the number of patients and testing of assays simultaneously in the same biopsy further validated their utility. A panel approach also improved testing outcome. A total of 14 UC progressors was readily separable from 15 UC nonprogressors and 6 normal controls. Chromosomal entropy (ie, the extent of alteration diversity) proved to be the most useful test. By receiver-operating characteristic analysis, mean chromosomal entropy in 28 patients over all four chromosomes yielded 100% sensitivity and 92% specificity for distinguishing progressors from nonprogressors with optimum choice of threshold. Moreover, separation was achieved using only nondysplastic and predominantly rectal (82.8%) biopsies that were remote from neoplasia, suggesting that full colonoscopy with extensive biopsies might be avoided for the majority of UC patients, the nonprogressors. These data further strengthen the concept that genomic biomarkers can distinguish UC progressors from nonprogressors and improve cancer surveillance in UC.
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Colitis Ulcerosa , Neoplasias Colorrectales , Marcadores Genéticos , Lesiones Precancerosas , Adolescente , Adulto , Anciano , Niño , Preescolar , Colitis Ulcerosa/genética , Colitis Ulcerosa/patología , Colon/patología , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/patología , Progresión de la Enfermedad , Femenino , Humanos , Hibridación Fluorescente in Situ , Lactante , Mucosa Intestinal/patología , Masculino , Persona de Mediana Edad , Lesiones Precancerosas/genética , Lesiones Precancerosas/patología , Curva ROC , Sensibilidad y Especificidad , Telómero/patología , Adulto JovenRESUMEN
Telomeres are repetitive DNA sequences at the end of each chromosome that provide stability and prevent end-to-end chromosome fusions. In order to understand mechanisms responsible for telomere shortening, it is necessary to develop methods for accurate telomere length measurement that can be applied to archival and fresh tissue and cells. This unit describes in situ-based quantitative fluorescence in situ hybridization (QFISH) protocols using a fluorescence-conjugated telomere probe (peptide nucleic acid, PNA) that stains telomeres proportionally to their length. These protocols can be used on formalin-fixed paraffin-embedded tissue, lightly fixed tissue, cells isolated from tissue, cultured cells, and agar-embedded cells. The basic protocol for QFISH staining is modified to achieve excellent QFISH staining for a variety of cell preparations. Image-analysis techniques to quantitate average telomere lengths from tissues and isolated stained cells are also described.
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Hibridación in Situ/métodos , Telómero/ultraestructura , Animales , Células/citología , Células/ultraestructura , Cromosomas/fisiología , Cromosomas/ultraestructura , Formaldehído , Procesamiento de Imagen Asistido por Computador , Espectrometría de Fluorescencia/métodosRESUMEN
BACKGROUND: Telomeres are tandem repeated DNA sequences at the ends of every chromosome, which cap, stabilize, and prevent chromosome fusions and instability. Telomere regulation is an important mechanism in cellular proliferation and senescence in normal diploid and neoplastic cells. Quantitative methods to assess telomere lengths are essential to understanding how telomere dynamics play a role in these processes. METHODS: Telomere lengths have been conventionally measured using terminal restriction fragment (TRF), quantitative fluorescence in situ hybridization (QFISH), and flow FISH. In this study, we have applied QFISH to measure average telomere lengths in cultured cells and human tissues of the GI tract. Importantly, this method can be used to analyze telomere lengths in sections using confocal microscopy. We describe and compare three image analysis algorithms: a simple pixel histogram calculation of background corrected fluorescence, a telomere spot-finding method, and a background curve subtraction algorithm. RESULTS: Using normal human diploid fibroblasts (NHDF) either dropped on slides or sectioned after agar embedding, similar telomere length shortening is evident with increasing population doubling levels (PDLs), using peptide nucleic acid (PNA) and an N3'-P5'-phosphoamidate (PA) oligonucleotide probe for all three methods. Validation of these in situ telomere quantification methods showed excellent agreement with the commonly used telomere repeat fragment-Southern blot method. Telomere length reductions can also be demonstrated in tissue sections from histologically normal mucosa from patients with chronic ulcerative colitis (with dysplasia or cancer elsewhere in the colon), in colon adenomas, and in mucosal biopsies from patients with Barrett's esophagus. Both on slides and in tissue sections, the telomere spot-finding method has the greatest variability, while intra- and inter-biopsy variability in telomere length assessment using the other methods is relatively low. CONCLUSIONS: Accurate and reproducible telomere length measurements can be made in tissue sections using QFISH and confocal microscopy. The simplest methods proved the most reliable and make these methods readily accessible to many laboratories. The use of these methods will enhance the ability to measure telomere lengths in tissue samples and aid in the understanding of the role of telomere length in aging and disease.
Asunto(s)
Algoritmos , Hibridación Fluorescente in Situ/métodos , Telómero/metabolismo , Esófago de Barrett/patología , Biopsia , Línea Celular , Senescencia Celular , Centrómero/genética , Centrómero/metabolismo , Colitis Ulcerosa/patología , Colon/patología , Fibroblastos , Humanos , Microscopía Confocal , Sondas de Oligonucleótidos/análisis , Sondas de Oligonucleótidos/genética , Ácidos Nucleicos de Péptidos/análisis , Ácidos Nucleicos de Péptidos/genética , Telómero/genéticaRESUMEN
Ulcerative colitis, a chronic inflammatory disease of the colon, is associated with a high risk of colorectal carcinoma that is thought to develop through genomic instability. We considered that the rapid cell turnover and oxidative injury observed in ulcerative colitis might accelerate telomere shortening, thereby increasing the potential of chromosomal ends to fuse, resulting in cycles of chromatin bridge breakage and fusion and chromosomal instability associated with tumor cell progression. Here we have used quantitative fluorescence in situ hybridization to compare chromosomal aberrations and telomere shortening in non-dysplastic mucosa taken from individuals affected by ulcerative colitis, either with (UC progressors) or without (UC non-progressors) dysplasia or cancer. Losses, but not gains, of chromosomal arms and centromeres are highly correlated with telomere shortening. Chromosomal losses are greater and telomeres are shorter in biopsy samples from UC progressors than in those from UC non-progressors or control individuals without ulcerative colitis. A mechanistic link between telomere shortening and chromosomal instability is supported by a higher frequency of anaphase bridges--an intermediate in the breakage and fusion of chromatin bridges--in UC progressors than in UC non-progressors or control individuals. Our study shows that telomere length is correlated with chromosomal instability in a precursor of human cancer.