RESUMO
OBJECTIVE: Vascular co-option is a resistance mechanism to anti-angiogenic agents, but combinations of anti-vascular agents may overcome this resistance. We report a phase 1b and randomised phase 2 trial to determine the safety and efficacy of pazopanib with fosbretabulin. METHODS: Eligible patients had recurrent, epithelial ovarian cancer with a platinum-free interval (PFI) of 3 to 12 months. Patients were stratified according to PFI (>6 versus ≤6 months) and prior bevacizumab use. RESULTS: Twelve patients were treated in the phase 1b. Commonest grade ≥ 2 adverse events (AEs) were hypertension (100%), neutropenia (50%), fatigue (50%), vomiting (50%). There was one DLT (grade 3 fatigue). The recommended phase 2 dose level was fosbretabulin 54 mg/m2 on days 1, 8 and 15 and pazopanib 600 mg once daily (od), every 28 days, which was then compared to pazopanib 800 mg od in a randomised phase 2 trial. Twenty-one patients were randomised (1:1) in the phase 2 trial. In phase 1b and phase 2, four patients treated with pazopanib and fosbretabulin developed reversible, treatment-related cardiac AEs, leading to premature discontinuation of the study. In the phase 2 trial, the median PFS was 7.6 months (95% CI 4.1-not estimated) versus 3.7 months (95% CI 1.0-8.1) in favour of the experimental arm (HR 0.30, 95% CI 0.09-1.03, P = .06). CONCLUSIONS: It remains unclear whether pazopanib with with fosbretabulin is an efficacious regimen to treat epithelial ovarian cancer. Effective cardiac risk mitigation is needed to increase the tolerability and maximize patient safety in future trials.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Carcinoma Epitelial do Ovário/tratamento farmacológico , Neoplasias Ovarianas/tratamento farmacológico , Pirimidinas/uso terapêutico , Sulfonamidas/uso terapêutico , Inibidores da Angiogênese/administração & dosagem , Inibidores da Angiogênese/efeitos adversos , Inibidores da Angiogênese/uso terapêutico , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Carcinoma Epitelial do Ovário/sangue , Carcinoma Epitelial do Ovário/irrigação sanguínea , Cardiotoxicidade/etiologia , Relação Dose-Resposta a Droga , Feminino , Humanos , Indazóis , Recidiva Local de Neoplasia , Neovascularização Patológica/sangue , Neovascularização Patológica/tratamento farmacológico , Neoplasias Ovarianas/sangue , Neoplasias Ovarianas/irrigação sanguínea , Intervalo Livre de Progressão , Pirimidinas/efeitos adversos , Estilbenos/administração & dosagem , Estilbenos/efeitos adversos , Sulfonamidas/efeitos adversos , Taxa de SobrevidaRESUMO
AIMS: There is a need for predictive and surrogate response biomarkers to support treatment with antiangiogenic vascular endothelial growth factor (VEGF) inhibitors. We aimed to identify a minimally-invasive biomarker predicting benefit from cediranib pretreatment or early during treatment in patients with recurrent or metastatic cervical cancer. METHODS: Blood samples were collected before treatment, during treatment and upon disease progression where appropriate from patients enrolled in CIRCCa, a randomised phase II trial of carboplatin and paclitaxel with or without cediranib. Plasma concentrations of VEGF-A, VEGF-receptor 2, Ang1 and Tie2 were measured using multiplex enzyme-linked immunosorbent assay. Pretreatment and temporal changes of the biomarkers were investigated using proportional hazard regression and unsupervised clustering analysis. RESULTS: Samples (n = 556) from 52 patients were analysed. VEGF-receptor 2 (P = .0006) and Tie2 (P = .04) were downregulated following cediranib, while VEGF-A (P = .0025) was upregulated. High Eastern Cooperative Oncology Group performance status (P = .02, hazard ratio [HR] = 2.15, 95% confidence interval [CI] 1.13-4.09) and low pretreatment Tie2 concentrations (P = .003, HR = 0.57, 95%CI 0.39-0.83) were independent prognostic factors associated with reduced progression-free survival. Two patterns of changes in VEGF-A following cediranib were identified. Patients with elevated VEGF-A in the first 3 treatment cycles, regardless of magnitude, had reduced progression-free survival in the placebo arm but improved survival with the addition of cediranib (P = .019, HR = 0.13, 95% CI 0.02-0.71). CONCLUSION: Patterns of early elevation in plasma VEGF-A should be studied further as a potential biomarker to predict treatment benefit from cediranib.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Biomarcadores Tumorais/sangue , Recidiva Local de Neoplasia/tratamento farmacológico , Quinazolinas/uso terapêutico , Neoplasias do Colo do Útero/tratamento farmacológico , Fator A de Crescimento do Endotélio Vascular/sangue , Adulto , Idoso , Progressão da Doença , Monitoramento de Medicamentos , Feminino , Humanos , Pessoa de Meia-Idade , Recidiva Local de Neoplasia/sangue , Recidiva Local de Neoplasia/mortalidade , Recidiva Local de Neoplasia/patologia , Intervalo Livre de Progressão , Neoplasias do Colo do Útero/sangue , Neoplasias do Colo do Útero/mortalidade , Neoplasias do Colo do Útero/patologia , Adulto JovemRESUMO
BACKGROUND: The monocarboxylate transporter-1 (MCT1) represents a novel target in rational anticancer drug design while AZD3965 was developed as an inhibitor of this transporter and is undergoing Phase I clinical trials ( http://www.clinicaltrials.gov/show/NCT01791595 ). We describe the optimisation of an immunofluorescence (IF) method for determination of MCT1 and MCT4 in circulating tumour cells (CTC) as potential prognostic and predictive biomarkers of AZD3965 in cancer patients. METHODS: Antibody selectivity was investigated by western blotting (WB) in K562 and MDAMB231 cell lines acting as positive controls for MCT1 and MCT4 respectively and by flow cytometry also employing the control cell lines. Ability to detect MCT1 and MCT4 in CTC as a 4(th) channel marker utilising the Veridex™ CellSearch system was conducted in both human volunteer blood spiked with control cells and in samples collected from small cell lung cancer (SCLC) patients. RESULTS: Experimental conditions were established which yielded a 10-fold dynamic range (DR) for detection of MCT1 over MCT4 (antibody concentration 6.25 µg/mL; integration time 0.4 seconds) and a 5-fold DR of MCT4 over MCT 1 (8 µg/100 µL and 0.8 seconds). The IF method was sufficiently sensitive to detect both MCT1 and MCT4 in CTCs harvested from cancer patients. CONCLUSIONS: The first IF method has been developed and optimised for detection of MCT 1 and MCT4 in cancer patient CTC.
Assuntos
Transportadores de Ácidos Monocarboxílicos/biossíntese , Proteínas Musculares/biossíntese , Pirimidinonas/administração & dosagem , Carcinoma de Pequenas Células do Pulmão/genética , Simportadores/biossíntese , Tiofenos/administração & dosagem , Antineoplásicos/administração & dosagem , Linhagem Celular Tumoral , Ensaios Clínicos Fase I como Assunto , Imunofluorescência , Voluntários Saudáveis , Humanos , Transportadores de Ácidos Monocarboxílicos/sangue , Transportadores de Ácidos Monocarboxílicos/genética , Proteínas Musculares/sangue , Proteínas Musculares/genética , Células Neoplásicas Circulantes/patologia , Carcinoma de Pequenas Células do Pulmão/sangue , Carcinoma de Pequenas Células do Pulmão/patologia , Simportadores/sangue , Simportadores/genéticaRESUMO
The oral dipeptidyl peptidase 1 (DPP1) inhibitor AZD5248 showed aortic binding in a rat quantitative whole-body autoradiography (QWBA) study, and its development was terminated prior to human dosing. A mechanistic hypothesis for this finding was established invoking reactivity with aldehydes involved in the cross-linking of elastin, a major component of aortic tissue. This was tested by developing a simple aldehyde chemical reactivity assay and a novel in vitro competitive covalent binding assay. Results obtained with AZD5248, literature compounds, and close analogues of AZD5248 support the mechanistic hypothesis and provide validation for the use of these assays in a two tier screening approach to support lead optimization. The strengths and limitations of these assays are discussed.
Assuntos
Aorta/metabolismo , Compostos de Bifenilo/metabolismo , Catepsina C/antagonistas & inibidores , Inibidores de Proteases/metabolismo , Animais , Autorradiografia , Compostos de Bifenilo/química , Catepsina C/metabolismo , Microscopia Eletrônica , Inibidores de Proteases/química , Ratos , Ratos WistarRESUMO
BACKGROUND: Matrix metalloproteinases (MMPs) are a family of proteolytic enzymes involved in extracellular matrix (ECM) homeostasis. MMPs have been an attractive pharmacological target for a number of indications. However, development has been hampered by the propensity of compounds targeting these enzymes to cause connective-tissue pathologies. The broad-spectrum MMP-inhibitor (MMPi) AZM551248 has been shown to induce such effects in the dog. Histopathological changes were consistent with fibrodysplasia (FD), characterised by fibroblast proliferation and the deposition of collagen in the subcutaneous tissues. We conducted a time-course study administering 20mg/kg/day AZM551248 between 4 and 17 days. Cervical subcutaneous tissue and plasma were sampled during the time-course. miRNA expression profiles in subcutaneous skin specimens following the administration of AZM551248 were determined by high-throughput-sequencing. RESULTS: An increasing number of miRNAs were differentially expressed compared with vehicle treated control animals as the study progressed. Several of these were members of the miR-200 family and were significantly attenuated in response to MMPi. As the severity of FD increased at the later time-points, other miRNAs associated with TGFß synthesis and regulation of the acute inflammatory response were modulated. Evidence indicative of epithelial to mesenchymal transition was present at all study time points. Receiver operator curve (ROC) analysis revealed that miR-21 expression in the cervical subcutaneous tissue was a sensitive and specific biomarker of FD incidence. CONCLUSIONS: Our data reveal significant perturbations in canine skin miRNA expression in response to MMPi administration. Furthermore, we have identified dysregulated miRNAs that are associated with processes relevant to the key histopathological events of MMPi-induced FD.
Assuntos
Doenças do Tecido Conjuntivo/induzido quimicamente , Doenças do Tecido Conjuntivo/genética , Inibidores de Metaloproteinases de Matriz/efeitos adversos , Metaloproteinases da Matriz/metabolismo , MicroRNAs/genética , Piperazinas/efeitos adversos , Pele/patologia , Animais , Biomarcadores/sangue , Colágeno Tipo III/sangue , Doenças do Tecido Conjuntivo/sangue , Doenças do Tecido Conjuntivo/patologia , Progressão da Doença , Cães , Perfilação da Expressão Gênica , Segurança , Tela Subcutânea/efeitos dos fármacos , Tela Subcutânea/metabolismo , Tela Subcutânea/patologia , Fatores de TempoRESUMO
Susceptibility to abacavir hypersensitivity has been attributed to possession of the specific human leukocyte antigen allele HLA-B*57:01. HLA-B*57:01-restricted activation of CD8+ T-cells provides a link between the genetic association and the iatrogenic disease. The objectives of this study were to characterize the functionality of drug-responsive CD8+ T-cell clones generated from HLA-B*57:01+ drug-naive subjects and to explore the relationship between abacavir accumulation in antigen presenting cells and the T-cell response. Seventy-four CD8+ clones expressing different Vß receptors were shown to proliferate and kill target cells via different mechanisms when exposed to abacavir. Certain clones were activated with abacavir in the absence of antigen presenting cells. Analysis of the remaining clones revealed two pathways of drug-dependent T-cell activation. Overnight incubation of antigen presenting cells with abacavir, followed by repeated washing to remove soluble drug, activated approximately 50% of the clones, and the response was blocked by glutaraldehyde fixation. In contrast, a 1 h antigen presenting cell pulse did not activate any of the clones. Accumulation of abacavir in antigen presenting cells was rapid (less than 1 h), and the intracellular concentrations were maintained for 16 h. However, intracellular abacavir was not detectable by mass spectrometry after pulsing. These data suggest that T-cells can be activated by abacavir through a direct interaction with surface and intracellular major histocompatibility complex (MHC) molecules. With the former, abacavir seemingly participates in the MHC T-cell receptor binding interaction. In contrast, the latter pathway likely involves MHC binding peptides displayed as a consequence of abacavir exposure, but not abacavir itself.
Assuntos
Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/efeitos dos fármacos , Didesoxinucleosídeos/farmacologia , Antígenos HLA-B/imunologia , Ativação Linfocitária/efeitos dos fármacos , Linfócitos T CD8-Positivos/imunologia , Morte Celular/efeitos dos fármacos , Linhagem Celular , Proliferação de Células , Células Clonais/citologia , Células Clonais/efeitos dos fármacos , Células Clonais/imunologia , Citocinas/imunologia , Didesoxinucleosídeos/química , Relação Dose-Resposta a Droga , Humanos , Leucócitos Mononucleares/citologia , Leucócitos Mononucleares/imunologia , Relação Estrutura-AtividadeRESUMO
Human exposure to abacavir, a primary alcohol antiretroviral, is associated with the development of immunological drug reactions in individuals carrying the HLA risk allele B*57:01. Interaction of abacavir with antigen presenting cells results in cell activation through an Hsp70-mediated Toll-like receptor pathway and the provision of T-cell antigenic determinants. Abacavir's electrophilic aldehyde metabolites are potential precursors of neoantigens. Herein, we have used mass spectrometry to study the oxidative metabolism of abacavir in EBV-transformed human B-cells. RNA and protein were isolated from the cells and subjected to transcriptomic and mass spectrometric analyses to identify the redox enzymes expressed. Low levels of isomeric abacavir carboxylic acids were detected in subcellular fractions of EBV-transformed human B-cells incubated with abacavir. Metabolite formation was time-dependent but was not reduced by an inhibitor of Class I alcohol dehydrogenases. Relatively high levels of mRNA were detected for several redox enzymes, including alcohol dehydrogenase 5 (Class III), aldehyde dehydrogenases (ALDH3A2, ALDH6A1, and ALDH9A1), CYP1B1, CYP2R1, CYP7B1, and hydroxysteroid dehydrogenase 10. Over 2600 proteins were identified by mass spectrometry. More than 1000 of these proteins exhibited catalytic activity, and 80 were oxido-reductases. This is the first proteomic inventory of enzymes in antigen presenting cells. However, neither of the hepatic alcohol dehydrogenases of Class I which metabolize abacavir in vitro was expressed at the protein level. Nevertheless the metabolic production of abacavir carboxylic acids by B-cell fractions implies abacavir-treated immune cells might be exposed to the drug's protein-reactive aldehyde metabolites in vivo.
Assuntos
Células Apresentadoras de Antígenos/citologia , Células Apresentadoras de Antígenos/metabolismo , Didesoxinucleosídeos/metabolismo , Biotransformação , Linhagem Celular Transformada , Citosol/metabolismo , Didesoxinucleosídeos/química , Humanos , Cinética , Fígado/citologia , Espectrometria de Massas , Conformação Molecular , Oxirredução , Frações Subcelulares/química , Frações Subcelulares/metabolismo , Fatores de TempoRESUMO
Tesaglitazar was developed as a dual peroxisome proliferator-activated receptor (PPARα/γ). To support the clinical program, a hamster carcinogenicity study was performed. The only neoplastic findings possibly related to treatment with tesaglitazar were low incidences of hemangioma and hemangiosarcoma in the liver of male animals. A high-power, two-year investigative study with interim necropsies was performed to further elucidate these findings. Treatment with tesaglitazar resulted in changes typical for exaggerated PPARα pharmacology in rodents, such as hepatocellular hypertrophy and hepatocellular carcinoma, but not an increased frequency of hemangiosarcomas. At the highest dose level, there was an increased incidence of sinusoidal dilatation and hemangiomas. No increased endothelial cell (EC) proliferation was detected in vivo, which was confirmed by in vitro administration to ECs. Immunohistochemistry and gene expression analyses indicated increased cellular stress and vascular endothelial growth factor (VEGF) expression in the liver, which may have contributed to the sinusoidal dilatation. A two-fold increase in the level of circulating VEGF was detected in the hamster at all dose levels, whereas no effect on VEGF was observed in patients treated with tesaglitazar. In conclusion, investigations have demonstrated that tesaglitazar does not produce hemangiosarcomas in hamster despite a slight effect on vascular morphology in the liver.
Assuntos
Alcanossulfonatos/toxicidade , Neoplasias Hepáticas Experimentais/induzido quimicamente , PPAR alfa/agonistas , PPAR gama/agonistas , Fenilpropionatos/toxicidade , Animais , Área Sob a Curva , Testes de Carcinogenicidade , Proliferação de Células/efeitos dos fármacos , Cricetinae , Feminino , Perfilação da Expressão Gênica , Hemangioma/induzido quimicamente , Hemangiossarcoma/induzido quimicamente , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Humanos , Imuno-Histoquímica , Fígado/efeitos dos fármacos , Fígado/metabolismo , Fígado/patologia , Masculino , Estatísticas não Paramétricas , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
UNLABELLED: Drug-induced liver injury (DILI) due to flucloxacillin is a rare but serious complication of treatment. There is some evidence that flucloxacillin is a human pregnane X receptor (PXR) agonist. This study was designed to investigate the relevance of PXR to flucloxacillin toxicity and to identify genes changing in expression in response to flucloxacillin. Changes in gene expression in human hepatocytes after treatment with 500 microM flucloxacillin for 72 hours were examined by expression microarray analysis. The ability of flucloxacillin to act as a PXR agonist was investigated with reporter gene experiments. Flucloxacillin DILI cases (n = 51), drug-exposed controls without toxicity (n = 64), and community controls (n = 90) were genotyped for three common PXR polymorphisms. Luciferase reporter assays were used to assess the significance of a promoter region PXR polymorphism. Seventy-two probe sets representing 50 different genes showed significant changes in expression of 1.2-fold or higher. Most genes showing changes greater than 3-fold were known to be rifampicin-responsive, and this suggested a PXR-dependent mode of regulation. Using a luciferase-everted repeat separated by 6 base pairs element construct, we confirmed that flucloxacillin was a PXR agonist. We found a difference in the distribution of a PXR polymorphism (rs3814055; C-25385T) between flucloxacillin DILI cases and controls with the CC genotype associated with an increased risk of disease (odds ratio = 3.37, 95% confidence interval = 1.55-7.30, P = 0.0023). Reporter gene experiments showed lower promoter activity for the C allele than the T allele. CONCLUSION: Flucloxacillin is a PXR agonist at pharmacologically relevant concentrations, and a functionally significant upstream PXR polymorphism is a risk factor for flucloxacillin-induced DILI.
Assuntos
Doença Hepática Induzida por Substâncias e Drogas/etiologia , Floxacilina/efeitos adversos , Receptores de Esteroides/fisiologia , Células Hep G2 , Humanos , Análise de Sequência com Séries de Oligonucleotídeos , Receptor de Pregnano X , Receptores de Esteroides/agonistas , Receptores de Esteroides/genética , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMO
Approximately 50% of patients with early-stage non-small-cell lung cancer (NSCLC) who undergo surgery with curative intent will relapse within 5 years1,2. Detection of circulating tumor cells (CTCs) at the time of surgery may represent a tool to identify patients at higher risk of recurrence for whom more frequent monitoring is advised. Here we asked whether CellSearch-detected pulmonary venous CTCs (PV-CTCs) at surgical resection of early-stage NSCLC represent subclones responsible for subsequent disease relapse. PV-CTCs were detected in 48% of 100 patients enrolled into the TRACERx study3, were associated with lung-cancer-specific relapse and remained an independent predictor of relapse in multivariate analysis adjusted for tumor stage. In a case study, genomic profiling of single PV-CTCs collected at surgery revealed higher mutation overlap with metastasis detected 10 months later (91%) than with the primary tumor (79%), suggesting that early-disseminating PV-CTCs were responsible for disease relapse. Together, PV-CTC enumeration and genomic profiling highlight the potential of PV-CTCs as early predictors of NSCLC recurrence after surgery. However, the limited sensitivity of PV-CTCs in predicting relapse suggests that further studies using a larger, independent cohort are warranted to confirm and better define the potential clinical utility of PV-CTCs in early-stage NSCLC.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/diagnóstico , Recidiva Local de Neoplasia/diagnóstico , Células Neoplásicas Circulantes/patologia , Veias Pulmonares/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/genética , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/patologia , Carcinoma Pulmonar de Células não Pequenas/cirurgia , Feminino , Regulação Neoplásica da Expressão Gênica/genética , Genoma Humano/genética , Humanos , Masculino , Pessoa de Meia-Idade , Metástase Neoplásica , Recidiva Local de Neoplasia/genética , Recidiva Local de Neoplasia/patologia , Recidiva Local de Neoplasia/cirurgia , Estadiamento de NeoplasiasRESUMO
The development of the dual peroxisome proliferator-activated receptor (PPAR) alpha/gamma agonist tesaglitazar as an oral antidiabetic was recently discontinued. Here we present tumor data from a 2-year carcinogenicity study in rats given 0.3, 1, 3, and 10 micromol/kg tesaglitazar is presented with focus on the findings of subcutaneous fibrosarcomas. To investigate the mechanism for induction of fibrosarcomas, replicative DNA synthesis (immunohistochemical detection of BrdU-labeled cells) and expression of PPARgamma (immunohistochemistry and reverse transcription-polymerase chain reaction) in subcutaneous adipose tissues was assessed in rats administered 1 or 10 micromol/kg for 2 weeks or 3 months. Poorly differentiated subcutaneous mesenchymal sarcomas with a predominant spindle cell appearance occurred at the highest dose level of 10 micromol/kg in both sexes, and these tumors were diagnosed as fibrosarcomas. The 10-micromol/kg dose was at or above the maximum tolerated dose and caused considerable cardiovascular mortality. Tesaglitazar stimulated DNA synthesis mainly in subcutaneous interstitial mesenchymal cells. The percentage of BrdU-labeled interstitial cells was increased at 1 and 10 micromol/kg after 2 weeks. The increase in DNA synthesis was still significant at the end of the 12-week treatment at 10 mumol/kg, the dose producing fibrosarcoma. However, at 1 micromol/kg, a dose below the no-observed-effect level for fibrosarcoma, the level of DNA synthesis was similar to control levels at 12 weeks. Immunohistochemical analyses showed no detectable PPARgamma protein in the majority of BrdU-labeled interstitial mesenchymal cells in white and brown fat. This indicates that stimulation of DNA synthesis is not mediated via direct activation of PPARgamma in these cells. The results suggest that the induction of rat fibrosarcoma by tesaglitazar, at exposures 100-fold above the human therapeutic exposure, may involve proliferation of undifferentiated mesenchymal cells in subcutaneous tissues.
Assuntos
Alcanossulfonatos/farmacologia , DNA/biossíntese , Fibrossarcoma/induzido quimicamente , Hipoglicemiantes/farmacologia , Mesoderma/metabolismo , PPAR alfa/agonistas , PPAR gama/agonistas , Fenilpropionatos/farmacologia , Neoplasias Cutâneas/induzido quimicamente , Animais , Antimetabólitos , Bromodesoxiuridina , Colesterol/sangue , Replicação do DNA/efeitos dos fármacos , Feminino , Fibrossarcoma/patologia , Imuno-Histoquímica , Masculino , Mesoderma/efeitos dos fármacos , Microdissecção , Tamanho do Órgão/efeitos dos fármacos , Ligação Proteica/efeitos dos fármacos , RNA/biossíntese , Ratos , Ratos Wistar , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Neoplasias Cutâneas/patologia , Triglicerídeos/sangueRESUMO
Tumor recurrence after surgical resection of NSCLC obstructs long-term disease-free survival in approximately 50% of cases. Our data suggest that combining circulating tumor cell enumeration (as single cells or clusters) in tumor-draining pulmonary vein and peripheral blood (assessed by CellSearch) at the time of NSCLC surgery better identifies those patients at higher risk for lung cancer recurrence than does peripheral circulating tumor cell number alone.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/cirurgia , Neoplasias Pulmonares/cirurgia , Células Neoplásicas Circulantes/metabolismo , Veias Pulmonares/patologia , Idoso , Carcinoma Pulmonar de Células não Pequenas/mortalidade , Carcinoma Pulmonar de Células não Pequenas/patologia , Feminino , Humanos , Neoplasias Pulmonares/mortalidade , Neoplasias Pulmonares/patologia , Masculino , Recidiva Local de Neoplasia , Células Neoplásicas Circulantes/patologia , Estudos Prospectivos , Taxa de SobrevidaRESUMO
Toxicogenomics is the application of gene expression profiling technology to toxicology. This results in the generation of very large and complex gene expression data sets associated with the development of toxicities. It is widely assumed that this data can be deconvoluted to reveal novel insights into toxicological processes that are of value to the task of risk assessment. More specifically, it is hoped that toxicogenomics will aid in the prediction of the toxic potential and mechanisms of toxicity of novel chemical entities. On the basis of such promise, the pharmaceutical industry has invested heavily in this area, as the perceived rewards in terms of improved pipeline efficiency and safer drugs are immense. Consequently, a great deal of groundwork has been done over the past several years to establish working methods in toxicogenomics, both within industry and academia, demonstrating utility in proof-of-concept studies, generating the databases on which some approaches depend, and developing new data analysis tools. Despite such activity, there is little reported evidence to suggest that toxicogenomics is making a significant impact on the discovery and development of drugs. This may partly reflect the understandable reluctance of pharmaceutical industries to share information in a competitive environment. It may also partly reflect difficulties in bridging the gap between theory and practice, as is required to deliver real value to the industry. This review will assess the successes and shortcomings of toxicogenomics, and consider how it can be usefully applied to a drug discovery pipeline.
Assuntos
Avaliação Pré-Clínica de Medicamentos/métodos , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica/efeitos dos fármacos , Animais , Desenho de Fármacos , Regulação da Expressão Gênica/genética , Humanos , Tecnologia Farmacêutica/economia , Tecnologia Farmacêutica/métodos , Tecnologia Farmacêutica/normasRESUMO
PURPOSE: Our aims are to determine levels of circulating cellular and protein biomarkers in hepatocellular carcinoma (HCC) patients and to analyse any relationships with clinical parameters. METHODS: Fifty-four consenting patients were recruited. Circulating tumour cells (CTCs) were enumerated (by CellSearch) and characterised via filtration [by isolation by size of epithelial tumour cells (ISET)] with downstream immunohistochemistry (IHC). Glypican-3 (GPC3) expression in tumour biopsies and CTCs (by IHC) was compared, and levels of circulating caspase-cleaved and full-length cytokeratin 18 (CK18, measured using M30 and M65 ELISAs) were examined as a putative prognostic factor and marker of tumour burden. RESULTS: CTCs were identified in 14 out of 50 (28%) patients by CellSearch and in 19 out of 19 (100%) patients by ISET. The presence of GPC3-positive CTCs by ISET was 100% concordant with the presence of GPC3-positive cells in the original tumour (n = 5). No statistically significant correlations were observed between CTC number and clinical characteristics, although trends were noted between CTC subtypes, Child-Pugh score and tumour node metastasis stage. Serum M30 and M65 levels (as continuous variables) significantly correlated with overall survival (OS) in a univariate analysis (p = 0.003 and p < 0.001, respectively); M65 levels remained statistically significant in a multivariate analysis (p = 0.029). CONCLUSIONS: This is the first study to detect GPC3-positive CTCs in HCC, important for drug development with this target. The significant association of circulating CK18 with OS in HCC further exemplifies the utility of circulating biomarkers in cancer.
Assuntos
Biomarcadores Tumorais/sangue , Carcinoma Hepatocelular/diagnóstico , Neoplasias Hepáticas/diagnóstico , Células Neoplásicas Circulantes/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Carcinoma Hepatocelular/sangue , Separação Celular , Feminino , Seguimentos , Glipicanas/sangue , Humanos , Técnicas Imunoenzimáticas , Queratina-18/sangue , Neoplasias Hepáticas/sangue , Masculino , Pessoa de Meia-Idade , Estadiamento de Neoplasias , PrognósticoRESUMO
Many efficacious cancer treatments cause significant cardiac morbidity, yet biomarkers or functional indices of early damage, which would allow monitoring and intervention, are lacking. In this study, we have utilized a rat model of progressive doxorubicin (DOX)-induced cardiomyopathy, applying multiple approaches, including cardiac magnetic resonance imaging (MRI), to provide the most comprehensive characterization to date of the timecourse of serological, pathological, and functional events underlying this toxicity. Hannover Wistar rats were dosed with 1.25 mg/kg DOX weekly for 8 weeks followed by a 4 week off-dosing "recovery" period. Electron microscopy of the myocardium revealed subcellular degeneration and marked mitochondrial changes after a single dose. Histopathological analysis revealed progressive cardiomyocyte degeneration, hypertrophy/cytomegaly, and extensive vacuolation after two doses. Extensive replacement fibrosis (quantified by Sirius red staining) developed during the off-dosing period. Functional indices assessed by cardiac MRI (including left ventricular ejection fraction (LVEF), cardiac output, and E/A ratio) declined progressively, reaching statistical significance after two doses and culminating in "clinical" LV dysfunction by 12 weeks. Significant increases in peak myocardial contrast enhancement and serological cardiac troponin I (cTnI) emerged after eight doses, importantly preceding the LVEF decline to <50%. Troponin I levels positively correlated with delayed and peak gadolinium contrast enhancement, histopathological grading, and diastolic dysfunction. In summary, subcellular cardiomyocyte degeneration was the earliest marker, followed by progressive functional decline and histopathological manifestations. Myocardial contrast enhancement and elevations in cTnI occurred later. However, all indices predated "clinical" LV dysfunction and thus warrant further evaluation as predictive biomarkers.
Assuntos
Antibióticos Antineoplásicos/toxicidade , Cardiomiopatias/patologia , Doxorrubicina/toxicidade , Miocárdio/ultraestrutura , Troponina I/sangue , Animais , Biomarcadores/sangue , Cardiomiopatias/sangue , Cardiomiopatias/induzido quimicamente , Cardiotoxicidade , Modelos Animais de Doenças , Fibrose , Testes de Função Cardíaca , Imageamento por Ressonância Magnética , Masculino , Ratos WistarRESUMO
Adverse drug reactions represent a major stumbling block to drug development and those with an immune etiology are the most difficult to predict. We have developed an in vitro T-cell priming culture method using peripheral blood from healthy volunteers to assess the allergenic potential of drugs. The drug metabolite nitroso sulfamethoxazole (SMX-NO) was used as a model drug allergen to establish optimum assay conditions. Naive T cells were cocultured with monocyte-derived dendritic cells at a ratio of 25:1 in the presence of the drug for a period of 8 days, to expand the number of drug-responsive T cells. The T cells were then incubated with fresh dendritic cells, and drug and their antigen responsiveness analyzed using readouts for proliferation, cytokine secretion, and cell phenotype. All five volunteers showed dose-dependent proliferation as measured by 5-(and 6)-carboxyfluorescein diacetate succinimidyl ester content and by (3)H-thymidine uptake. CD4 T cells that had divided in the presence of SMX-NO had changed from a naive phenotype (CD45RA+) to a memory phenotype (CD45RO+). These memory T cells expressed the chemokine receptors CCR2, CCR4, and CXCR3 suggesting a mixture of T(H)1 and T(H)2 cells in the responding population, with a propensity for homing to the skin. Drug stimulation was also associated with the secretion of a mixture of T(H)1 cytokines (interferon γ) and T(H)2 cytokines (interleukin [IL]-5 and IL-13) as detected by ELISpot. We are currently developing this approach to investigate the allergenic potential of other drugs, including those where an association between specific human leucocyte antigen alleles and susceptibility to an immunological reaction has been established.
Assuntos
Alérgenos/toxicidade , Técnicas de Cultura de Células , Avaliação Pré-Clínica de Medicamentos/métodos , Linfócitos T/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Técnicas de Cocultura , Citocinas/metabolismo , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/metabolismo , Relação Dose-Resposta a Droga , Humanos , Memória Imunológica/imunologia , Antígenos Comuns de Leucócito/metabolismo , Compostos Nitrosos/toxicidade , Receptores de Quimiocinas/metabolismo , Sulfametoxazol/toxicidade , Linfócitos T/imunologia , Linfócitos T/metabolismo , Linfócitos T Auxiliares-Indutores/efeitos dos fármacos , Linfócitos T Auxiliares-Indutores/imunologiaRESUMO
Matrix metalloproteinase (MMP) inhibitors, candidate therapeutic agents for a number of diseases, are known to be associated with acute fibrosis-type adverse effects in a number of species, including humans. The broad-spectrum MMP inhibitor, AZM551248, has previously been shown to cause these effects in the dog. Changes were characterized by the abnormal and extensive proliferation of fibroblasts and the deposition of collagen particularly in the subcutaneous connective tissues (subcutis) and were termed fibrodysplasia (FD). We performed a time-course study in dogs using AZM551248 and sampled skin, subcutis, and plasma before and during the development of FD. Detailed histopathological analysis and global gene expression profiling were performed on the subcutaneous tissues. The gene expression analysis of the subcutis indicated that extracellular matrix (ECM) remodeling was initiated asymptomatically at or before the earliest time point, day 4, and this was associated with dysregulation of expression of a number of MMPs and proteolytic enzymes. At later time points, the FD became progressively more extensive and severe, and this was associated with gene expression changes characteristic of tissue fibrosis, for example those associated with procollagen synthesis and processing. We postulate that AZM551248 inhibition of MMP action within the subcutis modulates the activity of several transcription factors and this in turn upregulates expression of specific proteases which initiate ECM remodeling. Persistent MMP inhibition results in the progression of ECM remodeling, culminating in collagen deposition and overt fibrosis. Our data indicate that inhibition of MMPs 1, 2, 3, and 9 is a key early event in AZM551248-induced FD in dog subcutis.
Assuntos
Matriz Extracelular/efeitos dos fármacos , Fibrose/induzido quimicamente , Inibidores de Metaloproteinases de Matriz , Piperazinas/toxicidade , Inibidores de Proteases/toxicidade , Pele/efeitos dos fármacos , Animais , Colágeno/metabolismo , Tecido Conjuntivo/efeitos dos fármacos , Tecido Conjuntivo/patologia , Modelos Animais de Doenças , Cães , Matriz Extracelular/metabolismo , Feminino , Fibrose/metabolismo , Fibrose/patologia , Expressão Gênica/efeitos dos fármacos , Perfilação da Expressão Gênica , Estudo de Associação Genômica Ampla , Metaloproteinases da Matriz/genética , Metaloproteinases da Matriz/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase em Tempo Real , Pele/patologiaRESUMO
Transcript profiling ("Transcriptomics") is a widely used technique that obtains information on the abundance of multiple mRNA transcripts within a biological sample simultaneously. Therefore, when a number of such samples are analysed, as in a scientific experiment, large and complex data sets are gene-rated. Here, we describe the use of one method commonly used to generate transcriptomics data, namely the use of Affymetrix GeneChip microarrays. Data generated in transcriptomics experiments can be analysed using a multitude of approaches, but a common goal is to identify those transcripts whose abundance is altered by the experimental conditions, or which differ between sets of samples. Here, we describe a simple approach, the calculation of the volcano score, which identifies transcripts with altered abundance, taking into account both the magnitude of the alteration and its statistical significance.
Assuntos
Perfilação da Expressão Gênica/métodos , Estatística como Assunto/métodos , Biotina/metabolismo , Células Cultivadas , DNA Complementar/biossíntese , DNA Complementar/genética , Humanos , Hibridização de Ácido Nucleico , Análise de Sequência com Séries de Oligonucleotídeos , Controle de Qualidade , RNA Complementar/biossíntese , RNA Complementar/genética , RNA Complementar/isolamento & purificação , RNA Complementar/metabolismo , Software , EspectrofotometriaRESUMO
Polyploidisation in hepatocytes has been associated with many physiologic and pathologic processes such as proliferation, metabolism, regeneration, aging, and cancer. We studied gene expression patterns in hepatocytes of different ploidy. Primary hepatocytes were obtained from mice of different ages: young (4-6 weeks old), adult (8-10 weeks old), and older (22-24 weeks old). Diploid (2N), tetraploid (4N), and octoploid (8N) hepatocytes were isolated for studies using a high-density mouse genome microarray. No major changes of gene expression patterns between hepatocytes of different ploidy were found. Fifty genes were identified as differentially expressed in the diploid and tetraploid populations, but the changes were less than twofold either way. Four genes (Gas2, Igfbp2, Nr1i3, and Ccne2) were differentially expressed in tetraploid and octoploid cells. This was confirmed in two age groups, "adult" and "older," but once again the factors were less than twofold and the expressions of Gas2 and Igfbp2 were more different between age groups than between ploidy classes. Our results show that polyploid hepatocytes are stable and "normal" without aberrant gene expression, unlike what is thought for cancer cells. By contrast to megakaryocytes, hepatocyte polyploidisation is not a differentiation step associated with major changes in gene expression. Our data support the hypothesis that hepatocyte polyploidisation is a protective mechanism against oxidative stress that occurs via a controlled process throughout growth and aging where binucleation is important.